WO2021152467A1

WO2021152467A1 - Combinations comprising bh3-mimetic compounds and inhibitors of early stages of autophagy for use in the treatment of non-hodgkin's b-cell lymphomas and lymphoid and/or myeloid leukemias

Info

Publication number: WO2021152467A1
Application number: PCT/IB2021/050610
Authority: WO
Inventors: Vincenzo CIMINALE; Vittoria RAIMONDI; Francesco CICCARESE
Original assignee: Universita' Degli Studi Di Padova; Istituto Oncologico Veneto Iov-Irccs
Priority date: 2020-01-28
Filing date: 2021-01-27
Publication date: 2021-08-05
Also published as: IT202000001567A1

Abstract

The present invention relates to combinations comprising BH3-mimetic compounds and at early stages authophagy inhibitors and/or pharmaceutically acceptable salts or derivatives thereof, pharmaceutical compositions comprising such combinations and use in the treatment of non-Hodgkin's B-cell lymphomas and lymphoid and/ or myeloid leukemias. In particular, the present invention relates to combinations comprising the BH3-mimetic compounds ABT-199 (venetoclax) and at least one at early stages authophagy inhibitor selected from SAR405 and Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof, or ABT-263 (navithoclax) and at least one at early stages authophagy inhibitor selected from V ps34-IN 1, SAR405 and Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof, to pharmaceutical compositions comprising such combinations, for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, even more preferably for the treatment of DLBCL, as well as in the treatment of lymphoid and/ or myeloid leukemias, preferably in the treatment of T- cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

Description

COMBINATIONS COMPRISING BH3-MIMETIC COMPOUNDS AND INHIBITORS OF EARLY STAGES OF AUTOPHAGY FOR USE IN THE TREATMENT OF NON-HODGKIN'S B-CELL LYMPHOMAS AND

LYMPHOID AND/OR MYELOID LEUKEMIAS

5

DESCRIPTION

TECHNICAL FIELD 0 The present invention relates to combinations comprising BH3-mimetic compounds and at early stages authophagy inhibitors and/or pharmaceutically acceptable salts or derivatives thereof, pharmaceutical compositions comprising such combinations and use in the treatment of non-Hodgkin's B-cell lymphomas and lymphoid and/ or myeloid leukemias. 5 More particularly, the present invention relates to pharmacological combinations comprising BH3-mimetic compounds and at least one at early stages authophagy inhibitor and/or pharmaceutically acceptable salts or derivatives thereof, to pharmaceutical compositions comprising such combinations and to the use of the combinations of the invention and of the compositions containing them for the treatment0 of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), marginal zone B-cell lymphoma or mantle cell lymphoma (MCL), and even more preferably for the treatment of DLBCL, as well as in the treatment of lymphoid and/ or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia5 (T-ALL) and acute myeloid leukemia (AML).

BACKGROUND

Escape from apoptosis is a strategy found in many types of cancer (Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell (2011) 144, 646-674, doi:10.1016/j.cell.2011.02.013), often involved in the resistance of cancer cells to0 chemotherapy.

One of the main mechanisms of apoptosis resistance is the overexpression of anti- apoptotic proteins belonging to the Bcl-2 family (Fernald, K. and Kurokawa, M. Evading apoptosis in cancer. Trends Cell (2013) Biol 23, 620-633, doi:10.1016/j.tcb.2013.07.006).

The components of the Bcl-2 family of proteins are the main regulators of mitochondrial- dependent apoptosis in eukaryotic cells and this family is composed of both anti- apoptotic (Bcl-XL, Bcl-2, Bcl-W, Bfl-1, MCL-1) and pro-apoptotic proteins (Bak, Bax, Bid, Bim, Bad, Bik, Bmf, Noxa, Puma).

In general, the Bcl-2 protein plays several roles, both physiological and pathological; among the most relevant are the inhibition of apoptosis, which, if altered, can promote aberrant cell survival. Therefore, the inhibition of Bcl-2 protein can reactivate cell death in pathological settings, leading to a better therapeutic response in cancer patients. In fact, in the prior art, the involvement of Bcl-2 proteins in a large number of solid and haematological neoplasms has been described. In particular, Bcl-2, which plays a central role in the survival of B lymphocytes, is expressed aberrantly in non-Hodgkin's B-cell lymphomas (neoplasms resulting from B-cell transformation) (Leverson, J.D. and Cojocari, D. Hematologic Tumor Cell Resistance to the BCL-2 Inhibitor Venetoclax: A Product of Its Microenvironment? Front. Oncol. (2018) vol. 8:458. doi: 10.3389/ fonc.2018.00458).

Non-Hodgkin's B-cell lymphomas (NHL) are haematological malignancies that primarily involve lymphoid tissues. Depending on the type, NHLs can be characterized by a painless development or by high aggressiveness. The term "non-Hodgkin's B-cell lymphomas" comprises a number of neoplastic diseases derived from B-cell transformation. The 5 most common types, in order of prevalence, are diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), marginal zone B-cell lymphoma and mantle cell lymphoma (MCL).

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of paediatric acute lymphoblastic leukemias and 25% of adult leukemias (Chiaretti, S. and Foa, R. T-cell acute lymphoblastic leukemia. Haematologica (2009) 94(2), 160-162, doi:

10.3324/haematol.2008.004150). It is a very aggressive tumour caused by the immature form of T lymphocytes (lymphoblasts), with onset in the thymus and rapid spread throughout the body. In patients, the spread of cancer cells to the central nervous system is often observed.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults (about 80% of cases). AML may occur in patients with pre-existing haematological disorders as a result of therapy or, in most cases, de novo, with abnormal proliferation of myeloid cells and monoclonal evolution of the disease. Although progress in AML treatment has led to significant improvements in the treatment of younger patients, prognosis in patients aged 65 years and older, representing the majority of new cases, remains inauspicious (De Kouchkovsky, I. and Abdul-Hay, M. Acute myeloid leukemia: a comprehensive review and 2016 update. Blood Cancer J (2016) 6(7):e441. doi: 10.1038/bcj.2016.50).

On the basis of these observations new anticancer drugs have been developed capable of inducing apoptosis in tumor cells, having the proteins of the Bcl-2 family as target.

BH3-mimetics are small molecules capable of antagonizing anti-apoptotic proteins by structurally mimicking the BH3 domain of pro-apoptotic proteins.

One of these BH3-mimetic molecules is venetoclax (also known as ABT-199, whose formula is given below), which selectively antagonizes the Bcl-2 protein (Souers, A. J. et al. ABT-199, a potent and selective Bcl-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. (2013) vol. 19, 202-208, doi:10.1038/nm.3048), leading to permeabilization of the external mitochondrial membrane and to the release of cytochrome c which activates the cell death programme for apoptosis.

IUPAC nomenclature 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexene-l-yl]methyl]piperazin- l-yl]-N-[3-nitro-4-(oxan-4-ylmethylamino)phenyl]sulfonyl-2-(lH-pyrrolo[2,3-b]pyridin- 5-yloxy)benzamide.

Although the use of ABT-199 has dramatically improved the prognosis of relapsing/ refractory chronic lymphocytic leukemia (RR-CLL, a form of non-Hodgkin's B-cell lymphoma), the antitumor activity thereof is highly variable in several haematological tumors, resulting, for example, very promising in the treatment of mantle cell lymphoma (MCL) but ineffective in other non-Hodgkin lymphomas, including diffuse large B-cell lymphoma (DLBCL) (Roberts, A. W. and Huang, D. Targeting BCL2 With BH3 Mimetics: Basic Science and Clinical Application of Venetoclax in Chronic Lymphocytic Leukemia and Related B Cell Malignancies. Clin Pharmacol Ther (2017) vol. 101, 89-98, doi:10.1002/ cpt.553).

In the case of T-cell acute lymphoblastic leukemia (T-ALL), ABT-199 has been tested exclusively on cell lines (in vitro and in vivo) and ex vivo on primary cells isolated from patients. Most T-ALL cell lines have been shown to be highly resistant to ABT-199 treatment. In particular, in this pathology ABT-199 is effective only in respect of immature cells, whereas it is ineffective in mature T-ALL (which constitute about 40% of clinical cases) (Peirs, S. et al. ABT-199 mediated inhibition of BCL-2 as a novel therapeutic strategy in T-cell acute lymphoblastic leukemia. Blood (2014) vol. 25, 3738-3747, doi: 10.1182/blood-2014-05-574566).

A phase 2 clinical study demonstrated that ABT-199 in monotherapy resulted in complete remission in only 19% of cases of acute myeloid leukemia (AML) previously treated with other therapies (Konopleva, M. et al. Efficacy and biological correlates of response in a phase II study of venetoclax monotherapy in patients with acute myelogenous leukemia. Cancer Discov (2016) vol. 10, 1106-1117, doi: 10.1158/2159-8290.CD-16-0313), indicating a high resistance of this disease to the treatment with ABT-199.

Therefore, the development of new therapies is extremely important for the treatment of patients with refractory neoplasms.

The Bcl-2 protein is not only involved in the control of apoptosis: Bcl-2, in fact, also resides on the surface of the endoplasmic reticulum, where it sequestrates the Beclin-1 protein, responsible for the formation of autophagosomes, thus suppressing authophagy. By eliminating damaged organelles and proteins, authophagy plays a key role in maintaining cell homeostasis and contributes to the resistance to anticancer therapies (Pei, G. et al. Autophagy Facilitates Metadherin-Induced Chemotherapy Resistance Through the AMPK/ ATG5 Pathway in Gastric Cancer. Cell Physiol Biochem (2018) vol. 46, 847-859, doi:10.1159/ 000488742).

Apoptosis and authophagy appear, therefore, to be highly interconnected mechanisms (Rubinstein, A. D. and Kimchi, A. Life in the balance - a mechanistic view of the crosstalk between autophagy and apoptosis. / Cell Sci (2012) vol. 125, 5259-5268, doi:10.1242/jcs.H5865), therefore it is reasonable to assume that ABT-199, in addition to releasing pro-apoptotic proteins at the mitochondrial level, can also trigger authophagy by breaking the Bcl-2 binding with Beclin-1. In the tumor context, authophagy has been described as a mechanism that favours both death and cell survival (Dalby, K. N., Tekedereli, L, Lopez-Berestein, G. and Ozpolat, B. Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Autophagy (2010) vol. 6, 322-329, doi:10.4161/auto.6.3.11625).

Since DLBCL, the most common type of non-Hodgkin's B-cell lymphoma, T-ALL and AML are neoplasms characterized by strong intrinsic resistance to ABT-199, treatment of patients with these diseases is severely limited in clinical practice compared to neoplasms such as CLL, therefore, the development of a new strategy to overcome cancer cell resistance to antitumor therapy with ABT-199 is extremely important.

In addition, a need is particularly felt for a reduction in the doses of ABT-199 necessary to achieve a therapeutic effect, in order to reduce the toxic effects of this active substance, which in particular manifest themselves as neutropenia, nausea, anaemia, diarrhoea, upper respiratory tract infection, fatigue, thrombocytopenia, male infertility. In particular, the toxicity of ABT-199 is reported also against normal B lymphocytes, which represent the healthy counterpart of DLBCL. This feature of ABT-199 requires a strategy that allows to widen the therapeutic window thereof so as to eliminate tumor cells using low doses of drug.

Treatment regimens based on the combination of standard chemotherapy (R-CHOP) and ibrutinib have been proposed for the treatment of DLBCL, which, while showing significant progress, did not enable to decrease the proportion of about 40% of patients with DLBCL that is currently incurable. Treatment of T-ALL involves a combination of corticosteroids (dexamethasone or prednisone), vincristine, L-asparaginase and an anthracycline (daunorubicin or doxorubicin). Although this therapy leads to complete remission in a high proportion of paediatric patients, about 20% of cases do not respond or have relapses and are therefore currently incurable. Treatment of AML involves two stages, induction of remission and maintenance, with chemotherapy based on cytarabine, daunorubicin and etoposide, or arsenic trioxide and all-trans retinoic acid. Targeted therapies are also available for the treatment of this disease. However, 50% of elderly patients (over 65 years of age) cannot be treated with intensive chemotherapy due to toxicity, resulting in an inauspicious prognosis (only 25% of patients survive 5 years after diagnosis).

Therefore, considering that approximately 40% of patients with DLBCL, 20% of T-ALL patients and 50% of elderly AML patients do not respond to currently available therapies and due to the high intrinsic resistance of these diseases to ABT-199, new therapies should be developed for patients with these refractory haematological malignancies. OBJECTS AND SUMMARY OF THE INVENTION

An object of the present invention is to overcome the disadvantages of the prior art.

In particular, it is an object of the present invention to provide a novel and inventive combination of pharmaceutical active ingredients for the treatment of non-Hodgkin's B- cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large cell B- lymphoma (DLBCL), as well as in the treatment of lymphoid and/ or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

These and other objects of the present invention are achieved by combining the BH3- mimetic compounds selected from ABT-199 (venetoclax) or ABT-263 (navithoclax) and at least one at early stages authophagy inhibitor and/ or pharmaceutically acceptable salts or derivatives thereof, selected from Vps34-IN1 and/ or pharmaceutically acceptable salts or derivatives thereof, SAR405 and/ or pharmaceutically acceptable salts or derivatives thereof or Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof, and by means of pharmaceutical compositions comprising such combinations together with one or more physiologically acceptable excipients.

The chemical structure of ABT-263 (navithoclax) and the IUPAC nomenclature thereof are reported below:

Navitoclax - ABT-263 ABT-263 (navitoclax): IUPAC nomenclature 4-[4-[[2-(4-chlorophenyl)-5,5- dimethylcyclohexene-l-yl]methyl]piperazin-l-yl]-N-[4-[[(2R)-4-morpholin-4-yl-l- phenylsulfanilbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl] sulfonylbenzamide.

ABT-263 is a BH3-mimetic compound that inhibits anti-apoptotic proteins Bcl-2, Bcl-XL and BCL-W. By virtue of this characteristic, in addition to showing high antitumor efficacy in monotherapy or in combination with standard chemotherapy in different types of cancer (TSE, C. et al. ABT-263: A Potent and Orally Bioavailable Bcl-2 Family Inhibitor. Cancer Res (2008) vol. 68, 3421-3428, doi:10.1158/0008-5472.CAN-07-5836), ABT-263 causes thrombocytopenia in treated patients, since anti-apoptotic protein Bcl- XL plays a key role in platelet survival (Zhang, H. et al. Bcl-2 family proteins are essential for platelet survival. Cell Death Differ (2007) vol. 14, 943-951, doi:10.1038/ sj.cdd.4402081). In order to overcome this important adverse effect, ABT-263 has been chemically modified to reduce the specificity thereof to the anti-apoptotic protein Bcl-2 alone, which is dispensable for platelet survival (Debrincat, M.A. et al. BCL-2 is dispensable for thrombopoiesis and platelet survival. Cell Death Dis (2015) vol. 6, el721, doi: 10.1038/cddis.2015.97), thus giving rise to the BH3-mimetic compound ABT-199. Nevertheless, the wide inhibitory spectrum of ABT-263 makes it effective even in ABT- 199-resistant tumors that show Bcl-XL overexpression. Currently, several clinical trials are underway to assess the safety and the efficacy of this compound as monotherapy or in combination with chemotherapy in haematological and solid tumors.

The chemical structure of Vps34-IN1 and the IUPAC nomenclature thereof are reported below:

Vps34-IN1: IUPAC nomenclature l-[[5-[2-[(2-chloropyridine-4-yl)amino]pyrimidin-4- yl]-4-(cyclopropylmethyl)pyrimidin-2-yl]amino]-2-methylpropan-2-ol.

The chemical structure of SAR405 and the IUPAC nomenclature thereof are reported below:

SAR405: Nomenclature IUPAC (S)-9-((5-chloropyridine-3-yl)methyl)-2-((R)-3- methylmorpholino)-8-(trifluoromethyl)-6,7,8,9-tetrahydro-4H-pyrimidine[l,2- a]pyrimidin-4-one.

The chemical structure of Compound 19 and the IUPAC nomenclature thereof are reported below:

Compound 19: IUPAC nomenclature l-[[4-(cyclopropylmethyl)-5-[2-(pyridin-4- ylamino)pyrimidin-4-yl]pyrimidin-2-yl]amino]-2-methylpropan-2-ol

The invention relates to combinations comprising the BH3-mimetic compounds ABT-199 (venetoclax) or ABT-263 (navithoclax) and at least one at early stages authophagy inhibitor and/or pharmaceutically acceptable salts or derivatives thereof, and pharmaceutical compositions comprising such combinations, for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/ or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML), according to the characteristics of the appended claims, which form an integral part of the present description.

The at least one at early stages authophagy inhibitor and/ or pharmaceutically acceptable salts or derivatives thereof is preferably Vps34-IN1 and/ or pharmaceutically acceptable salts or derivatives thereof, SAR405 and/or pharmaceutically acceptable salts or derivatives thereof, and/ or Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof.

Another aspect of the present invention proposes a combination comprising ABT-199 (venetoclax) and SAR405 and/or pharmaceutically acceptable salts or derivatives thereof, and pharmaceutical compositions comprising such a combination together with one or more physiologically acceptable excipients.

This aspect also relates to the combination comprising ABT-199 (venetoclax) and SAR405 and/ or pharmaceutically acceptable salts or derivatives thereof and the pharmaceutical compositions comprising it, for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of large B-cell diffuse lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

Another aspect of the present invention proposes a combination comprising ABT-199 (venetoclax) and Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof, and pharmaceutical compositions comprising such a combination together with one or more physiologically acceptable excipients.

This aspect also relates to the combination comprising ABT-199 (venetoclax) and Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof and the pharmaceutical compositions comprising it, for use in the treatment of non-Hodgkin's B- cell lymphomas, preferably for the treatment of large B-cell diffuse lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

Another aspect of the present invention proposes a combination comprising ABT-263 (navithoclax) and Vps34-IN1 and/ or pharmaceutically acceptable salts or derivatives thereof, and pharmaceutical compositions comprising such a combination together with one or more physiologically acceptable excipients.

This aspect also relates to the combination comprising ABT-263 (navithoclax) and Vps34- IN1 and/or pharmaceutically acceptable salts or derivatives thereof and the pharmaceutical compositions comprising it, for use in the treatment of non-Hodgkin's B- cell lymphomas, preferably for the treatment of large B-cell diffuse lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

Another aspect of the present invention proposes a combination comprising ABT-263 (navithoclax) and SAR405 and/or pharmaceutically acceptable salts or derivatives thereof, and pharmaceutical compositions comprising such a combination together with one or more physiologically acceptable excipients.

This aspect also relates to the combination comprising ABT-263 (navithoclax) and SAR405 and/or pharmaceutically acceptable salts or derivatives thereof and the pharmaceutical compositions comprising it, for use in the treatment of non-Hodgkin's B- cell lymphomas, preferably for the treatment of large B-cell diffuse lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

Another aspect of the present invention proposes a combination comprising ABT-263 (navithoclax) and Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof, and pharmaceutical compositions comprising such a combination together with one or more physiologically acceptable excipients.

This aspect also relates to the combination comprising ABT-263 (navithoclax) and Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof and the pharmaceutical compositions comprising it, for use in the treatment of non-Hodgkin's B- cell lymphomas, preferably for the treatment of large B-cell diffuse lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

Further features and advantages of the present invention will be more apparent from the description of the accompanying drawings.

DEFINITIONS

Unless otherwise defined, all the terms of the art, notations and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this description belongs. In some cases, terms with commonly understood meanings are defined herein for clarity's sake and/ or ready reference; the insertion of such definitions in the present description must therefore not be interpreted as representative of a substantial difference with respect to what is generally understood in the art.

The term "physiologically acceptable excipient" refers to a substance which lacks any specific pharmacological effect and which does not produce adverse reactions when administered to a mammal, preferably a human being. Physiologically acceptable excipients are well known in the art and are described, for example, in the Handbook of pharmaceutical excipients, sixth-edition (2009), herein incorporated for reference.

The term "pharmaceutically acceptable salts or derivatives" refers to those salts or derivatives which possess the biological efficacy and properties of the salified or derivatized compound and which do not produce adverse reactions when administered to a mammal, preferably a human being. The pharmaceutically acceptable salts may be inorganic or organic salts; examples of pharmaceutically acceptable salts include, but are not limited to: carbonate, hydrochloride, hydrobromide, sulfate, hydrogen sulfate, citrate, maleate, fumarate, trifluoroacetate, 2-naphthalenesulfonate, and para- toluenesulfonate. Further information on pharmaceutically acceptable salts can be found in the Handbook of pharmaceutical salts, P. Stahl C. Wermuth, WILEY-VCH, 127-133, 2008, incorporated herein for reference. Pharmaceutically acceptable derivatives include esters, ethers and N-oxides.

The terms "treat", "treating" and "treatment" refer to a method of alleviating or repealing a disease and/or the associated symptoms thereof, as well as to a method for administering drugs to induce a biological response.

The term "composition" as used herein is intended to comprise a product comprising the ingredients specified in the specified quantities, as well as any product resulting, directly or indirectly, from the combination of the ingredients specified in the specified quantities. By "pharmaceutically acceptable" it is meant that the vector, diluent or excipient must be compatible with the other components of the formulation and not deleterious to the recipient.

The terms "comprising", "having", "including" and "containing" are to be understood as open terms (i.e. the meaning "comprising, but not limited to") and are to be considered as a support also for terms such as "essentially consist of", "essentially consisting of", "to consist of" or "consisting of".

The term "at early stages authophagy inhibitors" refers to pharmacological compounds capable of inhibiting the formation of autophagosomes.

PIK3C3/Vps34 is an acronym for "phosphoinositide 3-kinase (PI3K) class III isoform/ vacuolar protein sorting 34", which refers to the phosphatidyllinositol 3-kinase enzyme, class III isoform, involved in the formation of phosphatidyllinositol 3- phosphate, a phospholipid needed for the generation of autophagosomes.

The term "autophagosomes" refers to vesicles incorporating intracellular materials (proteins or organelles) intended for degradation by authophagy.

The term "mitophagy refers to the degradation through authophagy of damaged or no longer necessary mitochondria.

"NHL" is an acronym for " non-Hodgkin lymphoma” .

The term "non-Hodgkin's B-cell lymphomas" refers to a series of neoplastic diseases derived from the transformation of B lymphocytes. The 5 most common types of non- Hodgkin's B-cell lymphomas are diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), marginal zone B-cell lymphoma and mantle cell lymphoma (MCL). "DLBCL" is an acronym for " diffuse large B-cell lymphoma” .

"CLL" is an acronym for " Chronic lymphocytic leukemia”.

"RR-CLL" is an acronym for refractory/ resistant chronic lymphocytic leukemia.

"R-CHOP" is an acronym where "R" denotes the active substance rituximab; "C" the active substance cyclophosphamide; "H" the active substance hydroxydaunorubicin, also known as doxorubicin; "O" the active substance Oncovin®, also known as vincristine; and "P" the active substance prednisone.

"T-ALL" is an acronym for "T-cell acute lymphoblastic leukemia”.

"AML" is an acronym for "Acute myeloid leukemia”.

"MCL" is an acronym for "mantle cell lymphoma”.

"PBMC" is an acronym for "peripheral blood mononuclear cells " which indicates the mononuclear fraction (lymphocytes and monocytes) of leukocytes isolated from peripheral blood.

"Bcl-2" is an acronym for "B-cell lymphoma-2 " which refers to a family of genes and their proteins with pro- or anti-apoptotic activity.

The term "CC50" stands for "Cytotoxic Concentration 50" which indicates the cytotoxic concentration of a compound capable of causing death of 50% of the cells constituting a cell population in vitro.

The term "CC30" stands for "Cytotoxic Concentration 30” which indicates the concentration of a compound capable of causing death of 30% of the cells constituting a cell population in vitro.

By the expression "specific cell death" it is meant the percentage of cells dying following a specific pharmacological treatment.

The expression "sensitizable cell lines" refers to cell lines which have become more susceptible to the cytotoxic action of the BH3-mimetic compounds ABT-199 or ABT-263, thanks to the combined treatment with at least one at early stages authophagy inhibitor, such as for example Vps34-IN1, SAR405 and/ or Compound 19.

By the expression "therapeutic window" it is meant the interval between the effective concentration and the toxic concentration of a drug treatment. This parameter defines the safety of the drug treatment itself in relation to the resulting clinical benefit.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described hereinafter with reference to some examples, given by way of non-limiting example, and illustrated in the appended drawings.

Figures 1A and IB show the demonstration that DLBCL, AML and T-ALL cell lines are resistant to ABT-199 and ABT-263. In Figure 1A the bars represent the CC50 values of ABT-199 in DLBCL, AML and T-ALL cell lines. The dashed line indicates the mean value of CC50 (1.9 Nm) reported in the literature for relapsing/refractory chronic lymphocytic leukemia (RR-CLL). In Figure IB, the bars represent the CC50 values of ABT-263 in DLBCL, AML and T-ALL cell lines.

In Figure 2, the curves indicate the specific cell death, measured by labelling with propidium iodide and cytofluorimetry, in thymocytes, PBMCs and B lymphocytes exposed to increasing concentrations of ABT-199 (1 Nm - 10 mM). Specific cell death was calculated as ((T-NT)/ (100-NT))^!Ί00, where T indicates the percentage of cells positive for labelling with propidium iodide (PI) and treated with drugs and NT indicates the percentage of cells positive for PI and treated with solvent.

In Figure 3, the ABT-199 induced autophagic flow was measured in cytofluorimetry using the CYTO-ID® Autophagy detection kit 2.0 (Enzo Life Sciences). Mean fluorescence intensities (MFI) were calculated on the measured value in the solvent-treated cells (DMSO). The treatment with chloroquine, used as a positive control, induces the accumulation of autophagosomes and, consequently, the increase of the MFI signal; this is the reference value for the induction of autophagic flow by ABT-199. These experiments were performed on DLBCL OCI Lyl9 cells.

Figures 4A, 4B, 4C, 4D, 4E and 4F show the effects of authophagy inhibition on tumor cells and on the corresponding healthy cells. DLBCL, AML and T-ALL cell lines were treated with chloroquine, Vps34-IN1, SAR405 and Compound 19; B lymphocytes, PBMC and thymocytes were treated with chloroquine, Vps34-IN1 and SAR405. The dose- response curves were calculated after 72 hours of treatment for tumor cells and after 48 hours of treatment for healthy cells, analysing specific cell death induced by chloroquine, Vps34-IN1, SAR405 and Compound 19 alone at different concentrations (10-100 mM for chloroquine; 0.25-5 pM for Vps34-IN1, SAR405, Compound 19).

In Figures 5 A, 5B and 5C it is indicated how inhibition of authophagy sensitizes cell lines of haematological tumors to ABT-199. The DLBCL, AML and T-ALL cell lines were treated with ABT-199, chloroquine, SAR405 and Compound 19. Dose-response curves were calculated after 72 hours of treatment, analysing ABT-199-induced specific cell death (1 Nm - 10 mM), alone (solid lines) or in combination with authophagy inhibitors (dashed lines). Specific cell death is calculated as described for Figure 2.

Figures 6a, 6B and 6C show the effects of combining ABT-199 with authophagy inhibitors in healthy cells. B lymphocytes, PBMC and thymocytes were treated with ABT-199, chloroquine and SAR405. Dose-response curves were calculated after 48 hours of treatment, analysing ABT-199-induced specific cell death (1 Nm - 10 pM), alone (solid lines) or in combination with authophagy inhibitors (dashed lines). Specific cell death is calculated as described for Figure 2.

Figures 7 A and 7B show how SAR405 and Compound 19 have a synergistic activity with ABT-199 in the induction of cell death. The isobolographic analysis was carried out in the DLBCL HBL1 cell line treated with ABT-199 (10, 25, 50 and 100 Nm) or SAR405/ Compound 19 at concentrations 1, 2.5, 5 and 10 pM, and with the combination of ABT-199 with SAR405/ Compound 19, maintaining a ratio of BH3-mimetic to authophagy inhibitor equal to 1:100. Combination index values are calculated using CompuSyn software, based on the Chou and Talalay algorithm (Chou, T. C. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res (2010) vol. 70, pages 440-446, doi:10.1158/0008-5472.can-09-1947).

In Figures 8A, 8B and 8C it is indicated how inhibition of authophagy sensitizes cell lines of haematological tumors to ABT-263. The DLBCL, AML and T-ALL cell lines were treated with ABT-263, Vps34-IN1, SAR405 and Compound 19. Dose-response curves were calculated after 48 hours of treatment, analysing ABT-263-induced specific cell death (1 Nm - 10 pM), alone (solid lines) or in combination with authophagy inhibitors (dashed lines). Specific cell death is calculated as described for Figure 2.

Figure 9 shows how silencing Beclin-1 and Vps34 increases ABT-199-induced cell death. The DLBCL HBL1 cell line was treated with Beclin-1 or Vps34 specific siRNA or with a control siRNA and treated with DMSO, ABT-199 (100 Nm and 1000 Nm) 24 hours after transfection. Specific cell death was measured in cytofluorimetry 48 hours after treatments, as described in Figure 2. The asterisks indicate a statistically significant difference between the indicated samples.

Figure 10 shows how electron microscopy analysis suggests the involvement of mitophagia in resistance to ABT-199. The DLBCL OCI Lyl9 line was treated with DMSO, ABT-199 1000 Nm, 50 pM chloroquine and with their combination, fixed in glutaraldehyde 24 hours later and analysed by means of transmission electron microscopy (TEM).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to combinations comprising the BH3-mimetic compounds ABT-199 or ABT-263 and at least one at early stages authophagy inhibitor and/or pharmaceutically acceptable salts or derivatives thereof, and to pharmaceutical compositions comprising such combinations; moreover, said combinations and pharmaceutical compositions are the object of the invention for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/ or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

Particularly preferred at early stages authophagy inhibitors and/or pharmaceutically acceptable salts or derivatives thereof are Vps34-IN1 and/ or pharmaceutically acceptable salts or derivatives thereof, SAR405 and/or pharmaceutically acceptable salts or derivatives thereof, and/ or Compound 19 and/ or pharmaceutically acceptable salts or derivatives thereof.

In particular, therefore, the invention relates to a combination comprising ABT-199 and SAR405 and/or pharmaceutically acceptable salts or derivatives thereof, and to the pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are the object of the invention for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

In a particularly preferred aspect, the invention relates to a combination comprising ABT- 199 and SAR405 and/ or pharmaceutically acceptable salts or derivatives thereof, and to pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are object of the invention for use in the treatment of diffuse large B-cell lymphoma, T-cell acute lymphoblastic leukemia and acute myeloid leukemia.

Furthermore, the invention relates to a combination comprising ABT-199 and Compound 19 and/or pharmaceutically acceptable salts or derivatives thereof, and to the pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are the object of the invention for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

According to another particularly preferred aspect, the invention relates to a combination comprising ABT-199 and Compound 19 and/or pharmaceutically acceptable salts or derivatives thereof, and to pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are the object of the invention for use in the treatment of diffuse large B-cell lymphoma, T-cell acute lymphoblastic leukemia and acute myeloid leukemia.

In particular, moreover, the invention relates to a combination comprising ABT-263 and Vps34-IN1 and/or pharmaceutically acceptable salts or derivatives thereof, and to the pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are the object of the invention for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

In a particularly preferred aspect, the invention relates to a combination comprising ABT- 263 and Vps34-IN1 and/or pharmaceutically acceptable salts or derivatives thereof, and to pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are the object of the invention for use in the treatment of diffuse large B-cell lymphoma, T-cell acute lymphoblastic leukemia and acute myeloid leukemia.

Furthermore, the invention relates to a combination comprising ABT-263 and SAR405 and/or pharmaceutically acceptable salts or derivatives thereof, and to the pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions are the object of the invention for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/ or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

According to another particularly preferred aspect, the invention relates to a combination comprising ABT-263 and SAR405 and/or pharmaceutically acceptable salts or derivatives thereof, and to pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are the object of the invention for use in the treatment of diffuse large B-cell lymphoma, T-cell acute lymphoblastic leukemia and acute myeloid leukemia.

Furthermore, the invention relates to a combination comprising ABT-263 and Compound 19 and/or pharmaceutically acceptable salts or derivatives thereof, and to the pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are the object of the invention for use in the treatment of non-Hodgkin's B-cell lymphomas, preferably for the treatment of diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma, and even more preferably for the treatment of diffuse large B-cell lymphoma, as well as in the treatment of lymphoid and/or myeloid leukemias, preferably in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

According to another particularly preferred aspect, the invention relates to a combination comprising ABT-263 and Compound 19 and/or pharmaceutically acceptable salts or derivatives thereof, and to pharmaceutical compositions comprising it; moreover, said combination and the pharmaceutical compositions thereof are the object of the invention for use in the treatment of diffuse large B-cell lymphoma, T-cell acute lymphoblastic leukemia and acute myeloid leukemia.

In fact, it has been surprisingly found that it is possible to sensitize DLBCL, AML and T- ALL cells, particularly DLBCL cells, to the action of ABT-199 and ABT-263 using at least one at early stages authophagy inhibitor, such as for example Vps34-IN1, SAR405, and/ or Compound 19. The results reported in the experimental section indicate that the clinical efficacy of ABT- 199 and ABT-263 against diffuse large B-cell lymphoma (DLBCL), T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) can be enhanced with specific authophagy inhibitors, such as SAR405 or Compound 19 (in association with ABT-199) and Vps34-IN1, SAR405 or Compound 19 (in association with ABT-263), which, unlike chloroquine, result to be safe for healthy PBMCs, T cells and B cells. This new pharmacological approach can therefore increase the therapeutic window, allowing the selective elimination of resistant cancer cells using lower and less toxic doses of ABT- 199 and ABT-263.

Pharmaceutical compositions for use in the present invention generally comprise an effective amount of the combination object of the invention and a suitable pharmaceutical acceptable vehicle. The compositions can be prepared in a per se known manner, which usually involves mixing the compounds of the combination of the invention with one or more pharmaceutically acceptable carriers and, if desired, in combination with other active pharmaceutical compounds, when necessary under aseptic conditions. Reference is made to standard manuals, such as the latest edition of Remington's pharmaceutical Sciences.

Generally, for pharmaceutical use, the compounds may be formulated as a pharmaceutical preparation comprising at least one compound and at least one pharmaceutically acceptable vehicle, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.

The pharmaceutical combinations of the present invention are preferably in a unit dosage form and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, phial or any other suitable single-dose or multidose support or container (which can be properly labelled); optionally with one or more leaflets containing product information and/ or instructions for use.

Formulations containing the combination described herein can be prepared using a pharmaceutically acceptable vehicle which is considered safe and effective and can be administered to an individual without causing undesirable biological effects or undesired interactions. As generally used in this document, the "vector" includes, but is not limited to, diluents, binders, lubricants, disintegrators, fillers, pH modifying agents, preservatives and solubility enhancers.

The vector also includes components of the coating composition which may include plasticizers, pigments, colourants, stabilizing agents and glidants. Delayed-release, sustained-release and/or pulsatile-release dosage formulations can be prepared as described in standard references such as "Pharmaceutical dosage forms; Tablets", Ed. Liberman et al. (New York, Marcel Dekker, Inc., 1989), "Remington - the Science and practice of Pharmacy", 20th edition (Lippincott Williams & Wilkins, Baltimore, Maryland, 2000) and "Pharmaceutical dosage forms and drug delivery systems", 6th edition, Ansel et al. (Media, PA: Williams and Wilkins, 1995). These references provide information on supports, materials, equipment and processes for the preparation of tablets and capsules and delayed-release dosage forms of tablets, capsules and granules.

The invention is described below by means of experimental examples, which are not to be considered limiting for the purpose of the invention.

EXAMPLES

EXAMPLE 1

Study of the behaviour of ABT-199 and ABT-263 in haematological malignancies.

In order to demonstrate that, in the context of haematological malignancies, the autophagic elimination of damaged organelles may favour the survival of tumor cells treated with ABT-199 or ABT-263, the response to these compounds was evaluated in a panel of five cell lines derived from DLBCL (OCI Lyl9, SUDHL6, SUDHL4, U2932 and HBL1), a cell line derived from T-cell acute lymphoblastic leukemia (T-ALL; Jurkat) and two cell lines derived from acute myeloid leukemia (AML; KG-1 and MOLM-13). These cell lines were seeded (lxlO⁶ cells/ ml culture medium) in 48-well cell culture plates and treated with concentrations of ABT-199 or ABT-263 equal to 1, 10, 100, 1000 and 10000 Nm, obtaining the values of CC50 shown in Figures 1 A and IB. The various concentrations of ABT-199 or ABT-263 were prepared in the dimethylsulfoxide (DMSO) vehicle at an initial concentration 1000 times higher than the final concentration, so that in all cases the solvent was diluted 1:1000 in the culture medium.

All the analysed cell lines have been shown to be relatively resistant to ABT-199 (Figure 1A) when compared with what is reported in the literature for RR-CLL (CC₅₀ 1.9 Nm, represented by the dashed line of Figure 1A) (Anderson, M. A. et al. The BCL2 selective inhibitor venetoclax induces rapid onset apoptosis of CLL cells in patients via a TP53- independent mechanism. Blood (2016) vol. 127, pages 3215-3224, doi:10.1182/blood-2016- 01-688796). Moreover, the same cell lines have been shown to be resistant to low doses of ABT-263. Peripheral blood mononuclear cells (PBMCs), B lymphocytes and healthy thymocytes were also seeded (lxlO⁶ cells/ml culture medium) in 48-well cell culture plates and treated with concentrations of ABT-199 equal to 1, 10, 100, 1000 and 10000 Nm, obtaining the specific cell death values shown in Figure 2. The various concentrations of ABT-199 were prepared in the dimethylsulfoxide (DMSO) vehicle at an initial concentration 1000 times higher than the final concentration, so that in all cases the solvent was diluted 1:1000 in the culture medium. Specific cell death (%) was measured by labelling with propidium iodide (PI) and cytofluorimetry and was calculated as ((T- NT)/ (100-NT))^!Ί00, where T indicates the percentage of cells treated with drugs positive for PI labelling and NT indicates the percentage of cells positive for PI labelling treated with solvent. The B lymphocytes used in these experiments were isolated from mononuclear leukocytes deriving from peripheral blood of healthy donors by immunomagnetic selection using the "B Cell Isolation Kit II, Human" kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions.

It is important to note that the healthy mononuclear cells from peripheral blood (PBMCs), the B lymphocytes and the thymocytes are more sensitive to ABT-199 than tumor cells (Figure 2), although they never reach CC50 (represented by the dotted line in Figure 2) at any dose of ABT-199. These results underline the importance of developing innovative therapeutic strategies that effectively hit cancer cells with doses of ABT-199 that are less toxic to normal cells.

In order to test the effects of ABT-199 on autophagy, a cytofluorimetric assay (CYTO-ID® Autophagy Detection Kit 2.0, Pnzo Life Sciences) based on the selective fluorescent detection of authophagic vacuoles, was used, thus allowing a rapid and quantitative monitoring of the autophagic flow in viable cells. Preliminary data obtained in OCI Lyl9 cells revealed that ABT-199 increases the accumulation of autophagosomes in the presence of chloroquine, a known inhibitor of late authophagy phases, compared to treatment with chloroquine alone (Figure 3). DLBCL OCI Lyl9 cells were seeded (lxlO⁶ cells/ ml culture medium) in 6-well cell culture plates and treated with DMSO, ABT-199 1000 Nm, 50 mM chloroquine and the combination of ABT-199 and chloroquine. ABT-199 was prepared in the dimethylsulfoxide (DMSO) vehicle, chloroquine was prepared in the H2O vehicle, both at an initial concentration 1000 times higher than the final concentration, so that in all cases the solvent was diluted 1:1000 in the culture medium. 24 hours after the treatments, the cells were harvested, washed, labelled with the specific fluorophore and analysed by means of the cytofluorimeter as indicated in the protocol provided by the kit manufacturer. The mean fluorescence intensity (MFI) of each sample was compared to that obtained for the sample treated with the solvent only (DMSO). The fact that ABT-199 alone does not lead to an increase in the fluorescent signal is probably due to the rapid degradation of autophagosomes through their fusion with lysosomes. However, by blocking the formation of autophagolysosomes and the degradation by lysosomal proteins using chloroquine, it is possible to appreciate the induction of the autophagic flux in the cells treated with ABT-199 through an increase in mean fluorescence intensity (MFI). It is important to note that, however, chloroquine treatment induces cell death in all tested tumor cell lines, as well as in healthy cells, in a dose- dependent manner.

EXAMPLE 2

Demonstration of the effects of at early stages authophagy inhibitors in DLBCL, AML and T-ALL cell lines.

In order to obviate the lack of selectivity of chloroquine, two more specific compounds, Vps34-IN1 and SAR405, were used, which inhibit the first stages of formation of the autophagosome. DLBCL, AML and T-ALL cell lines were seeded (lxlO⁶ cells/ ml culture medium) in 48-well cell culture plates and treated with increasing concentrations of Vps34-IN1 (0.25, 0.5, 1, 2, 5 mM), SAR405 (0.25, 0.5, 1, 2, 5 mM), Compound 19 (0.25, 0.5, 1, 2, 5 pM) or chloroquine (10, 25, 50, 75, 100 pM). The various concentrations of Vps34-IN1, SAR405 and Compound 19 were prepared in the solvent dimethylsulfoxide (DMSO), the various concentrations of chloroquine were prepared in the solvent LhO, all at an initial concentration 1000 times higher than the final concentration, so that in all cases the solvent was diluted 1:1000 in the culture medium. Specific cell death (%) was measured by labelling with propidium iodide (PI) and cytofluorimetry and was calculated as ((T- NT)/ (100-NT))^!Ί00, where T indicates the percentage of cells treated with drugs positive for PI labelling and NT indicates the percentage of cells positive for PI labelling treated with solvent.

Compared to chloroquine, these compounds appear less toxic as individual agents (Figure 4A, 4B, 4C). The various concentrations of Vps34-IN1, SAR405 and Compound 19 were prepared in the solvent dimethylsulfoxide (DMSO), chloroquine was prepared in the solvent LhO, all at an initial concentration 1000 times higher than the final concentration, so that in all cases the solvent was diluted 1:1000 in the culture medium. Specific cell death (%) was measured by labelling with propidium iodide (PI) and cytofluorimetry and was calculated as ((T-NT)/ (100-NT))^!Ί00, where T indicates the percentage of cells treated with drugs positive for PI labelling and NT indicates the percentage of cells positive for PI labelling treated with solvent.

Moreover, Vps34-IN1 and SAR405 administered as individual agents do not damage normal B lymphocytes, PBMCs and healthy thymocytes (Figure 4D, 4E, 4F). In contrast, as mentioned above, the treatment with chloroquine alone leads also to damage to healthy cells (Figure 4D, 4E, 4F). Normal B cells, PBMCs and healthy thymocytes were seeded (lxlO⁶ cells/ ml culture medium) in 48-well cell culture plates and treated with increasing concentrations of Vps34-IN1 (0.25, 0.5, 1, 2, 5 pM), SAR405 (0.25, 0.5, 1, 2, 5 pM) or chloroquine (10, 25, 50, 75, 100 pM).

EXAMPLE 3

Demonstration of overcoming ABT-199 resistance in DLBCL, AML and T-ALL cell lines using the combinations of ABT-199 with SAR405 or Compound 19.

It is interesting to note that the at early stages authophagy inhibitors produce an intense sensitization to ABT-199 in all the cell lines used (Figures 5A, 5B, 5C), in particular of DLBCL derivation. In line with what has been described above, DLBCL, AML and T-ALL cell lines were seeded (lxlO⁶ cells/ ml culture medium) in 48-well cell culture plates and treated with increasing concentrations of ABT-199 (1, 10, 100, 1000 and 10000 Nm) alone or in combination with SAR405 (1 mM), Compound 19 (1 mM) or chloroquine (50 pM).

Furthermore, SAR405 does not increase the cytotoxic effect of ABT-199 in B lymphocytes, PBMCs and thymocytes (Figure 6A, 6B, 6C), suggesting that the inhibition of the early stages of authophagy can drastically improve the clinical efficacy of ABT-199, possibly reducing the side effects thereof and allowing a reduction in dosage.

The isobolographic analysis carried out in DLBCL HBL1 cells using the Chou and Talalay equation (Chou, T. C. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res (2010) vol. 70, pages 440-446, doi:10.1158/0008- 5472.can-09-1947) revealed that the effects on cell death of ABT-199 in combination with SAR405 or Compound 19 are highly synergistic (Figure 7 A and 7B). In fact, these Figures report the combination index obtained in the DLBCL HBL1 cell line. The cells were seeded (lxlO⁶ cells/ ml culture medium) in 48-well cell culture plates and treated with concentrations of ABT-199 equal to 10, 25, 50 and 100 Nm, of SAR405 or Compound 19 equal to 1, 2.5, 5 and 10 pM, and with the combination of ABT-199 and SAR405/ Compound 19, maintaining a ratio between the two drug concentrations equal to 1:100. The various concentrations of ABT-199, SAR405 and Compound 19 were prepared in the solvent dimethylsulfoxide (DMSO), at an initial concentration 1000 times higher than the final concentration, so that in all cases the solvent was diluted 1:1000 in the culture medium.

EXAMPLE 4 Demonstration of overcoming ABT-263 resistance in DLBCL, AML and T-ALL cell lines using the combinations of ABT-263 with Vps34-IN1, SAR405 or Compound 19.

In line with what has been observed with ABT-199, the at early stages authophagy inhibitors produced intense sensitization to ABT-263 in all the cell lines used (Ligure 8A, 8B, 8C). In line with what has been described above, DLBCL, AML and T-ALL cell lines were seeded (lxlO⁶ cells/ ml culture medium) in 48-well cell culture plates and treated with increasing concentrations of ABT-263 (1, 10, 100, 1000 and 10000 nM) alone or in combination with Vps34-IN1 (1 mM), SAR405 (1 pM), Compound 19 (1 pM) or chloroquine (50 pM).

EXAMPLE 5

Demonstration through a genetic approach of overcoming ABT-199 resistance in DLBCL, AML and T-ALL cell lines.

To validate the results obtained in example 3 through a genetic approach, it was evaluated whether the silencing of Beclin-1 or Vps34, using siRNA, potentiated the cytotoxicity of ABT-199 in DLBCL HBL1 cells. The cells (5xl0⁶ cells /sample) were electroporated with control siRNA or Beclin-1 or Vps34 specific siRNA, using the Neon electroporator (Life Technologies) and the associated kit, according to the manufacturer's recommendations and with the following parameters: 1410 V, 30 msec, one pulse. After electroporation, cells were seeded in culture medium in 6-well cell culture plates and incubated at 37°C with a CO2 tension equal to 5%. After 24 hours, electroporated cells were treated with DMSO or ABT-199 100 Nm or 1000 Nm. 48 hours after treatment, specific cell death (%) was measured by labelling with propidium iodide (PI) and cytofluorimetry and was calculated as ((T-NT)/ (100-NT))*100, where T indicates the percentage of cells treated with drugs positive for PI labelling and NT indicates the percentage of cells positive for PI labelling treated with solvent. As expected, the silencing of Beclin-1 (and, to a lesser extent, also of Vps34) leads to an increase in ABT- 199-mediated cell death (Figure 9). Collectively, these data suggest that authophagy is responsible for mitigating the pro-apoptotic effects induced by ABT-199.

EXAMPLE 6

Study of the role that authophagy can play through the elimination of dysfunctional mitochondria.

To verify that authophagy can play a survival role through the elimination of dysfunctional mitochondria, the effect of ABT-199 on DLBCL OCI Lyl9 cells was examined by means of transmission electron microscopy (TEM). For this analysis, cells were seeded (lxlO⁶ cells/ ml culture medium for a total of 5xl0⁶ cells) in cell culture flasks and treated with DMSO, ABT-199 at a concentration of 1000 Nm, chloroquine at a concentration of 50 mM and with the combination of ABT-199 and chloroquine. ABT-199 was prepared in the solvent DMSO, chloroquine was prepared in the solvent EhO, both at an initial concentration 1000 times higher than the final concentration, so that in all cases the solvent was diluted 1:1000 in the culture medium. After 24 hours from treatment, the cells were fixed in glutaraldehyde for 30 minutes, washed and kept at 4°C in a 0.1 M cacodylate sodium-based solution at pH 7.4, until inclusion in epoxy resin, cutting into ultrafine sections and subsequent acquisition of images under the microscope. The results show how the mitochondria of cells treated with ABT-199 are dilated and more electron transparent than the mitochondria of vehicle treated cells and how the structure of the ridges is partially compromised. It is interesting to note that in the presence of chloroquine, capable of blocking the degradation of autophagosome, the cells treated with ABT-199 contain debris resembling mitochondria (Figure 10), suggesting that mitophagy may be involved in resistance to ABT-199.

The results reported above therefore indicate how the clinical efficacy of ABT-199 and ABT-263 against different haematological tumors can be enhanced with specific authophagy inhibitors, such as Vps34-IN1, SAR405 or Compound 19, which, unlike chloroquine, are safe for PBMCs, healthy T and B cells. This new pharmacological approach can therefore increase the therapeutic window, allowing the selective elimination of resistant cancer cells using lower and less toxic doses of ABT-199 and ABT-

Claims

1. A combination comprising the BH3-mimetic ABT-199 compound and an at early stages authophagy inhibitor selected from SAR405 and/ or pharmaceutically acceptable salts or derivatives thereof or Compound 19 and/or pharmaceutically acceptable salts or derivatives thereof.

2. The combination comprising the BH3-mimetic ABT-263 compound and an at early stages authophagy inhibitor selected from Vps34-IN1 and/or pharmaceutically acceptable salts or derivatives thereof, SAR405 and/ or pharmaceutically acceptable salts or derivatives thereof or Compound 19 and/or pharmaceutically acceptable salts or derivatives thereof.

3. A pharmaceutical formulation comprising any of the combinations according to any one of claims 1 to 2 and one or more physiologically acceptable excipients.

4. A combination according to any one of claims 1 to 2, for use in the treatment of non- Hodgkin's B-cell lymphomas.

5. The combination according to claim 4, wherein the non-Hodgkin lymphomas are selected from diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma.

6. The combination according to claim 5, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.

7. The combination according to any one of claims 1 to 2, for use in the treatment of lymphoid leukemias.

8. The combination according to claim 7, wherein lymphoid leukemia is T-cell acute lymphoblastic leukemia.

9. The combination according to any one of claims 1 to 2, for use in the treatment of myeloid leukemias.

10. The combination according to claim 9, wherein myeloid leukemia is acute myeloid leukemia.

11. A pharmaceutical formulation according to claim 3, for use in the treatment of non- Hodgkin's B-cell lymphomas.

12. The pharmaceutical formulation according to claim 11, wherein the non-Hodgkin lymphomas are selected from diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, marginal zone B-cell lymphoma or mantle cell lymphoma.

13. The pharmaceutical formulation according to claim 12, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.

14. The pharmaceutical formulation according to claim 3, for use in the treatment of lymphoid leukemias.

15. The pharmaceutical formulation according to claim 14, wherein the lymphoid leukemia is T-cell acute lymphoblastic leukemia.

16. The pharmaceutical formulation according to claim 3, for use in the treatment of myeloid leukemias.

17. The pharmaceutical formulation according to claim 16, wherein the myeloid leukemia is acute myeloid leukemia.