Combination therapy

Abstract

The present invention relates to novel combination therapies for the treatment of ALL. The combination therapies comprises the administration of a BCL2 inhibitor in combination with a CREBBP and / or EP300 inhibitor.

Description

COMBINATION THERAPY FIELD OF THE INVENTION

[0001] The present invention relates to novel combination therapy for the treatment of cancer. BACKGROUND OF THE INVENTION

[0002] Cancer is caused by uncontrolled and unregulated cellular proliferation. Precisely what causes a cell to become malignant and proliferate in an uncontrolled and unregulated manner has been the focus of intense research over recent decades. This research has led to the identification of a number of molecular targets and key metabolic pathways that are known to be associated with malignancy. Despite numerous advances in the treatment of cancer, there remains a need for new therapies that provide improved therapeutic outcomes.

[0003] There is a particular need for novel therapies for certain haematological cancers, such as lymphomas. B-ALL is an example of a particularly aggressive haematological malignancy of B lineage progenitors and is the commonest cancer in children. Whilst the majority of children can be cured with multi-agent chemotherapy, patients with high-risk genetic subtypes, certain age groups and those who relapse remain a clinical challenge, such that B-ALL remains a leading cause of death in childhood (1). Furthermore, outcomes of adults with B- ALL remain dismal, even when fit enough to be treated with intensive chemotherapy, CAR-T cell therapy and allogenic bone marrow transplantation (2). There is therefore a need to better understand drivers of high-risk B-ALL and to develop novel therapeutic approaches targeting these challenging patient cohorts.

[0004] CREBBP mutations are found in multiple hematological and solid malignancies, in particular B cell lymphomas (3, 4). Loss-of-function (LOF) mutations affecting CREBBP are recurrent second-hit mutation across multiple genetic subtypes of B-ALL and are associated with adverse features, including high-risk genetic subtypes and persistent measurable residual disease(5-8). In addition, they have been mechanistically associated with chemoresistance and are more frequently found in relapse (5, 9-13). CREBBP mutations have also been described as an adverse prognostic factor in ALL, AML and Follicular Lymphoma (14-16). CREBBP is a large protein with histone acetyltransferase (HAT) enzymatic activity alongside protein scaffolding function mediated through multiple protein-protein interaction domains, including a bromodomain responsible for binding acetylated lysine residues. Alongside its paralogue EP300, CREBBP is considered to primarily function as a transcriptional co- activator, responsible for laying down activating acetyl marks on key histone residues at gene enhancers and promoters, but is also responsible for acetylating diverse non-histone targets,such as TP53. CREBBP LOF mutations can include complete loss of the protein or recurrent point mutations affecting the HAT domain, which have been postulated to exert a stronger phenotype (5). During B-ALL evolution, CREBBP mutations frequently become bi-allelic and commonly co-associate with activating lesions of the RAS signalling pathway, suggesting a strong oncogenic co-operativity between these lesions (5, 13, 17).

[0005] The principal approach currently used to target cells harbouring LOF mutations in tumour suppressor genes (TSG) is to perturb “synthetic lethal” dependencies that are acquired upon loss of TSG activity, commonly via inhibition of parallel redundant pathways or protein paralogues. In the context of CREBBP, this has been particularity studied in models of B cell lymphoma through inhibition of residual EP300 function using selective small molecule HAT or bromodomain inhibitors (18). Global analysis of genetic co-dependencies in cancer have also implicated a potential dependency of CREBBP-mutated tumours on EXOC5 function (19), whilst a number of mechanistic studies have identified potentially targetable roles for CREBBP in modulating key cellular processes including DNA damage response, signalling, apoptosis and metabolism (4, 13, 17, 20).

[0006] In light of the above, there is a need to identify novel therapies to treat ALL, and B-ALL in particular. There is also a need for improved therapies to treat, CREBBP-mutated high-risk B-ALL.

[0007] The present invention was devised with the foregoing in mind. SUMMARY OF THE INVENTION

[0008] Data presented in the example section herein shows that the administration of BCL2 inhibitors in combination with a CREBBP and / or EP300 inhibitor resulted in a significant increase in the observed anti-tumour response in various B-ALL diease models.

[0009] The present invention relates to a combination comprising: a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof; and a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof;

[0010] In a further aspect, the present invention relates to a combination as defined herein for use in the treatment of acute lymphoblastic leukamia (ALL).

[0011] In another aspect the present invention relates to a pharmaceutical product for use in the treatment of acute lymphoblastic leukamia (ALL) comprising a combination as defined herein.

[0012] In another aspect, the present invention relates to a pharmaceutical composition for use in the treatment of acute lymphoblastic leukamia (ALL) comprising a combination as defined herein, and one or more pharmaceutically acceptable excipients.

[0013] In another aspect, the present invention relates to a combination, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein, for use in the treatment of B-cell acute lymphoblastic leukaemia (B-ALL).

[0014] In another aspect, the present invention relates to the use of a combination as defined herein in the manufacture of a medicament for the use in the treatment of acute lymphoblastic leukamia (ALL).

[0015] In another aspect, the present invention relates to the use of a combination as defined herein in the manufacture of a medicament for the treatment of B-cell acute lymphoblastic leukaemia (B-ALL).

[0016] In another aspect, the present invention relates to a method of treating acute lymphoblastic leukamia (ALL) in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a combination as defined herein.

[0017] In another aspect, the present invention relates to a method of treating B-cell acute lymphoblastic leukaemia (B-ALL) in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a combination as defined herein.

[0018] In another aspect, the present invention relates to a BCL-2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, for use in the treatment of acute lymphoblastic leukamia (ALL), wherein the BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, is for simultaneous, separate or sequential administration with a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptablle salt thereof.

[0019] In another aspect, the present invention relates to a BCL-2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, for use in the treatment of B-cell acute lymphoblastic leukaemia (B-ALL), wherein the BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, is for simultaneous, separate or sequential administration with a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptablle salt thereof.

[0020] In another aspect, the present invention relates to a CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof, for use in the treatment of acutelymphoblastic leukamia (ALL), wherein the CREBBP and / or EP300 inhibitor inhibitor is for simultaneous, separate or sequential administration with a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof.

[0021] In another aspect, the present invention relates to a CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof, for use in the treatment of B-cell acute lymphoblastic leukaemia (B-ALL), wherein the CREBBP and / or EP300 inhibitor inhibitor is for simultaneous, separate or sequential administration with a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof.

[0022] In another aspect, the present invention relates to the use of a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of acute lymphoblastic leukamia (ALL), wherein the medicament is for simultaneous, separate or sequential administration with a CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof.

[0023] In another aspect, the present invention relates to the use of a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of B-cell acute lymphoblastic leukaemia (B-ALL), wherein the medicament is for simultaneous, separate or sequential administration with a CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof.

[0024] In another aspect, the present invention relates to the use of a CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of acute lymphoblastic leukamia (ALL), wherein the medicament is for simultaneous, separate or sequential administration with a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof.

[0025] In another aspect, the present invention relates to the use of a CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of B-cell acute lymphoblastic leukaemia (B-ALL), wherein the medicament is for simultaneous, separate or sequential administration with a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof.

[0026] In another aspect, the present invention relates to a method of treating acute lymphoblastic leukamia (ALL) comprising adminstering to a subject in need thereof a therapeutically effective amount of a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, and a CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof, wherein the BCL2 inhibitor as defined herein, or a pharmaceuticallyacceptable salt thereof, and the CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof are administered sequentially, separately or simultaneously with one another.

[0027] In another aspect, the present invention relates to a method of treating B-cell acute lymphoblastic leukaemia (B-ALL) comprising adminstering to a subject in need thereof a therapeutically effective amount of a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, and a CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof, wherein the BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, and the CREBBP and / or EP300 inhibitor inhibitor, or a pharmaceutically acceptable salt thereof are administered sequentially, separately or simultaneously with one another.

[0028] Preferred, suitable, and optional features of any one particular aspect of the present invention described herein are also preferred, suitable, and optional features of any other aspect. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0029] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0030] It is to be appreciated that references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

[0031] A “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for thedisease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.

[0032] References to “ALL” herein refer to acute lymphoblastic leukaemia, which includes B- cell acute lymphoblastic leukaemia (B-ALL), T-cell acute lymphoblastic leukaemia (T-ALL), lymphoblastic lymphoma (a solid tumour variant of ALL) and relate to both adults and children including B cell progenitor ALL (BCP-ALL). References to “B-ALL” herein refer to B-cell acute lymphoblastic leukaemia (B-ALL) specifically.

[0033] An “inhibitor” may be a polypeptide, nucleic acid, carbohydrate, lipid, small molecular weight compound, a PROTAC, an oligonucleotide, an oligopeptide, siRNA, antisense, a recombinant protein, an antibody, a peptibody, or conjugates or fusion proteins thereof. For a review of siRNA see Milhavet O, Gary DS, Mattson MP. (Pharmacol Rev. 2003 Dec;55(4):629-48. For a review of antisense see Opalinska JB, Gewirtz AM. Sci STKE.2003 Oct 28; 2003 (206): p47. A small molecular weight compound refers to a compound with a molecular weight of less than 2000 Daltons, less than 1000 Daltons, less than 700 Daltons or less than 500 Daltons.

[0034] References to “a pharmaceutically acceptable salt” of an inhibitor defined herein is refers to any salt form suitable for pharmaceutical use. Examples of pharmaceutically acceptable salts include an acid-addition salt of an inhibitor of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoracetic, formic, citric methane sulfonate or maleic acid. In addition, a suitable pharmaceutically acceptable salt of an inhibitor of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

[0035] References herein to a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, and a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, include, where appropriate, any isomeric, tautomeric, polymorphic, amorphous and solvate (e.g. hydrate) forms of the inhibitors. An inhibitor may also be administered in the form of a prodrug which is broken down in the human or animal body to release the active inhibitor. Examples of pro-drugs include in vivo cleavable ester derivatives of the inhibitors that may be formed at a carboxy group or a hydroxy group in an inhibitor compound and in-vivo cleavable amide derivatives that may be formed at a carboxy group oran amino group in an inhibitor compound. Various forms of pro-drug have been described, for example in the following documents :- a) Methods in Enzymology, Vol.42, p.309-396, edited by K. Widder, et al. (Academic Press, 1985); b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and Application of Pro-drugs”, by H. Bundgaard p.113- 191 (1991); d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); f) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984); g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, A.C.S. Symposium Series, Volume 14; and h) E. Roche (editor), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987.

[0036] References herein to a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, being administered “in combination with” a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or vice versa, unless otherwise stated otherwise, include the inhibitors being administered sequentially, separately or simultaneously with one another.

[0037] As used herein “simultaneous administration” refers to therapy in which the both agents (e.g. a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, and CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof) are administered at the same time, suitably as a dual-therapy.

[0038] As used herein “sequential administration” means that one agent is administered after the other, however, the time period between the administration of each agent is such that both agents are capable of acting therapeutically concurrently. Thus, administration "sequentially" may permit one agent to be administered within seconds, minutes, or a matter of hours after the other provided the circulatory half-life of the first administered agent is such that they are both concurrently present in therapeutically effective amounts. The time delay between the administration of the agents may vary depending on the exact nature of the agents, the interaction there between, and their respective half-lives.

[0039] As used herein, "separate administration" means that one agent is administered after the other, however, the time period between administration is such that the first administered agent is no longer present a therapeutically effective amount when the second agent isadministered, but the physiological consequences of the administration of the first administered agent may still be ongoing. Accordingly, the two agents exert their therapeutic effects separately, but pre-treatment with the first agent enhances the activity of the second agent. In other words, the overall therapeutic effect observed when the two agents separately act therapeutically may be greater than either agent used alone.

[0040] As used herein the “subject(s)” and / or “patient(s)”, suitably refer to mammals (e.g. humans and non-human mammals such as livestock (cows, sheep, goats) or companion animals (cats, dogs, horses, rabbits). Suitably, the subject(s) and / or patient(s) are human(s).

[0041] As used herein, a “pharmaceutical product” refers to a product comprising a pharmaceutical. For instance, examples of a pharmaceutical product include a medical device, a pharmaceutical composition and a kit of parts suitably comprising one or more devices, containers and / or pharmaceuticals. Combination therapy - BCL2 inhibitors with CREBBP and / or EP300 inhibitors

[0042] The present invention resides in the recognition that BCL2 inhibitors, when administered in combination with a CREBBP and / or EP300 inhibitor, produced a synergistic increase in potency in disease models of B-ALL.

[0043] Thus, the administration of a BCL2 inhibitor synergistically enhances the antitumour effect of a CREBBP and / or EP300 inhibitor therapy and vice versa. The combination of a BCL2 inhibitor and a CREBBP and / or EP300 inhibitor therefore offers a promising therapy for ALL more generally and B-ALL in particular.

[0044] The combination treatment of the present invention has the potential to provide better therapeutic outcomes in cancer patients, especially cancer patients that do not respond well to therapy with a BCL2 inhibitor or a CREBBP and / or EP300 inhibitor alone. The BCL2 inhibitor

[0050] Any suitable BCL2 inhibitor may be used in this combination therapy of the present invention. Suitably, the BCL2 inhibitor is approved for therapeutic use by national and regional regulatory authorities, such as the Food and Drug Administraion in the United States.

[0051] The BCL2 inhibitor may be a polypeptide, nucleic acid, small molecular weight compound, a PROTAC, an oligonucleotide, an oligopeptide, siRNA, antisense oligonucleotide, a recombinant protein, an antibody, a peptibody, or conjugates or fusion proteins thereof.

[0052] In a particular embodiment, the BCL2 inhibitor is an antisense oligonecleotide (e.g. the antisense oligonucleotide oblimersen sodium, G3139). For a review of antisense see Opalinska JB, Gewirtz AM. Sci STKE.2003 Oct 28; 2003 (206): p47.

[0053] In a further embodiment, the BCL2 inhibitor is a small molecule compound that inhibits BCL2.

[0054] Suitably, the BCL2 inhibitor is selected from the group consisiting of venetoclax (ABT- 199), navitoclax (ABT-263), obatoclax (GX15-070), oblimersen sodium (G3139), HA14-1, sabutoclax, S55746 (S055746, BCL201), gambogic acid, lisaftoclax (APG2575), sonrotoclax (BGB-11417), AZD4320, AZD-0466 and AT-101, or pharmaceutically acceptable salts thereof.

[0055] The chemical name for ventoclax is 4-{4-[(4'-chloro-5,5-dimethyl[3,4,5,6- tetrahydro[1,1'-biphenyl]]-2-yl)methyl]piperazin-1-yl}-N-(3-nitro-4-{[(oxan-4- yl)methyl]amino}benzene-1-sulfonyl)-2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide, and its chemical structure is shown below:

[0056] The chemical name for navitoclax is 4-(4-{[2-(4-Chlorophenyl)-5,5-dimethylcyclohex- 1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phenylsulfanyl)butan-2- yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide, and its chemical structure is shown below:

[0057] The chemical name for obatoclax is 2-(2-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-3- methoxy-2H-pyrrol-5-yl)-1H-indole, and its chemical structure is shown below:

[0058] The chemical name for HA14-1 is 2-Amino-6-bromo-α-cyano-3-(ethoxycarbonyl)-4H- 1-benzopyran-4-acetic acid ethyl ester, and its chemical structure is shown below:

[0059] The chemical name for sabutoclax is 2,3,5-trihydroxy-7-methyl-N-[(2R)-2- phenylpropyl]-6-[1,6,7-trihydroxy-3-methyl-5-[[(2R)-2-phenylpropyl]carbamoyl]naphthalen-2- yl]naphthalene-1-carboxamide, and its chemical structure is shown below:

[0060] The chemical name for S55746 is (S)-N-(4-hydroxyphenyl)-3-(6-(3- (morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzo[d][1,3]dioxol-5-yl)-N- phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide, and its chemical structure is shown below:

[0061] The chemical name for gambogic acid is (2Z)-4-[(1R,3aS,5S,11R,14aS)-8-Hydroxy- 3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-11-(4-methylpent-3-en-1-yl)-7,15-dioxo-3a,4,5,7- tetrahydro-1H,3H,11H-1,5-methanofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl]-2-methylbut-2- enoic acid, and its chemical structure is shown below:

[0062] The chemical name for lisaftoclax (APG2575) is 4-[4-[[8-(4- Chlorophenyl)spiro[3.5]non-7-en-7-yl]methyl]piperazin-1-yl]-N-[4-[[(2S)-1,4-dioxan-2-yl]methylamino]-3-nitrophenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, and its structure is shown below:

[0063] The chemical structure for sonrotoclax (BGB-11417) is shown below:

[0064] AZD4320 is a dual BCL2 / BCLxL inhibitor with the chemical name 4-(4-((R)-(4'-chloro- [1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-4-((2- hydroxyethyl)(methyl)amino)-1-(phenylthio)butan-2-yl)amino)-3- ((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide, and the structure shown below:

[0065] AZD0466 is a dendrimer formed from AZD4320 conjugated via a linker to a poly-L- lysine dendrimer.

[0066] AT-101 is a BCL2 inhibitor with the chemical name R-(-)-gossypol acetic acid, the structure of which is shown below:

[0067] In a further embodiment, the BCL2 inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), obatoclax (GX15-070), or oblimersen sodium (G3139), or a pharmaceutically acceptable salt thereof.

[0068] In a further embodiment, the BCL2 inhibitor is selected from the group consisting of venetoclax (ABT-199) or navitoclax (ABT-263), or a pharmaceutically acceptable salt thereof.

[0069] In a particular embodiment, the BCL2 inhibitor is venetoclax (ABT-199), or a pharmaceutically acceptable salt thereof. The CREBBP and / or EP300 inhibitor

[0070] Any suitable CREBBP and / or EP300 inhibitor may be used in this combination therapy of the present invention. Suitably, the CREBBP and / or EP300 inhibitor is approved for therapeutic use in humans by national and regional regulatory authorities, such as the Food and Drug Administraion in the United States.

[0071] In an embodiment, the CREBBP and / or EP300 inhibitor is a selected from the group consitisting of a CREBBP specific inhibitor or a dual CREBBP and EP300 inhibitor. In a further embodiment, the CREBBP and / or EP300 inhibitor is a CREBBP specific inhibitor (i.e. it does not inhibit EP300). In another embodiment, the CREBBP and / or EP300 inhibitor is a dual inhibitor that inhibits both CREBBP and EP300.

[0072] The CREBBP and / or EP300 inhibitor may be a polypeptide, nucleic acid, small molecular weight compound, a PROTAC, an oligonucleotide, an oligopeptide, siRNA,antisense oligonucleotide, a recombinant protein, an antibody, a peptibody, or conjugates or fusion proteins thereof.

[0073] In a particular embodiment, the CREBBP and / or EP300 inhibitor is an antisense oligonecleotide.

[0074] In a particular embodiment, the CREBBP and / or EP300 inhibitor is a PROTAC degrader (e.g. JQAD1).

[0075] In a further embodiment, the CREBBP and / or EP300 inhibitor is a small molecule compound that inhibits CREBBP and / or EP300.

[0076] Suitably, the CREBBP and / or EP300 inhibitor is selected from the group consisting of inobrodib, A-485, EP31670, CBP30, GNE-781, JQAD1 or pharmaceutically acceptable salts thereof.

[0077] The chemical name for inobrodib is (6S)-1-(3,4-difluorophenyl)-6-[5-(3,5-dimethyl-1,2- oxazol-4-yl)-1-(4-methoxycyclohexyl)benzimidazol-2-yl]piperidin-2-one, and its chemical structure is shown below:

[0078] The chemical name for A485 is (1R)-N-[(4-Fluorophenyl)methyl]-2,3-dihydro-5- [[(methylamino)carbonyl]amino]-2',4'-dioxo-N-[(1S)-2,2,2-trifluoro-1-methylethyl]spiro[1H- indene-1,5'-oxazolidine]-3'-acetamide, and its chemical structure is shown below:

[0079] The chemical name for EP31670 is 1,3-dimethyl-5-[2-(oxan-4-yl)-1-[2- (trifluoromethoxy)ethyl]-1H-1,3-benzodiazol-6-yl]-1,2-dihydropyridin-2-one, and its chemical structure is shown below:

[0080] The chemical name for CBP30 is (S)-4-(1-(2-(3-chloro-4-methoxyphenethyl)-5-(3,5- dimethylisoxazol-4-yl)-1H-benzo[d]imidazol-1-yl)propan-2-yl)morpholine, and its chemical structure is shown below:

[0081] The chemical , pp.9162-9183) is 3-(7- (Difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-N-methyl-1- (tetrahydro-2H-pyran-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxamide, and its chemical structure is shown below:

[0082] The chemical name for JQAD1 is 12-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin- 5-yl)amino)-N-((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)- 2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidin]-5-yl)dodecanamide, and its chemical structure is shown below:

[0083] In a further embodiment, the CREBBP and / or EP300 inhibitor is selected from the group consisting of inobrodib, A-485, EP31670, or pharmaceutically acceptable salts thereof.

[0084] In a further embodiment, the CREBBP and / or EP300 inhibitor is selected from the group consisting of inobrodib or A-485, or pharmaceutically acceptable salts thereof.

[0085] In a particular embodiment, the CREBBP and / or EP300 inhibitor is inobrodib, or a pharmaceutically acceptable salt thereof. Therapeutic Uses

[0086] The present invention also relates to a combination as defined herein (i.e a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, and a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof), for use in: (i) the treatment of ALL; (ii) the treatment of B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia; (iii) the treatment of B-ALL, BCP-ALL or T-ALL; (iv) the treatment of B-ALL; (v) the treatment of BCP-ALL; (vi) the treatment of T-ALL; or (vii) the treatment of lymphoblastic leukaemia.

[0087] The present invention also provides a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a BCL2 inhibitor or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients, for use in (i) the treatment of ALL; (ii) the treatment of B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia; (iii) the treatment of B-ALL, BCP-ALL or T-ALL;(iv) the treatment of B-ALL; (v) the treatment of BCP-ALL; (vi) the treatment of T-ALL; or (vii) the treatment of lymphoblastic leukaemia; wherein the BCL2 inhibitor is administered in combination with a CREBBP and / or EP300 inhibitor.

[0088] In an embodiment, the BCL2 inhibitor is administered simultaneously, separately or sequentially with the CREBBP and / or EP300 inhibitor.

[0089] The present invention aslo provides a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a CREBBP and / or EP300 inhibitor or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients, for use in (i) the treatment of ALL; (ii) the treatment of B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia; (iii) the treatment of B-ALL, BCP-ALL or T-ALL; (iv) the treatment of B-ALL; (v) the treatment of BCP-ALL; (vi) the treatment of T-ALL; or (vii) the treatment of lymphoblastic leukaemia; wherein the CREBBP and / or EP300 inhibitor is administered in combination with a BCL2 inhibitor.

[0090] In an embodiment, the CREBBP and / or EP300 inhibitor is administered simultaneously, separately or sequentially with the BCL2 inhibitor.

[0091] The present invention also provides the use of a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a BCL2 inhibitor or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients, in the manufacture of a medicament for use in (i) the treatment of ALL; (ii) the treatment of B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia; (iii) the treatment of B-ALL, BCP-ALL or T-ALL; (iv) the treatment of B-ALL; (v) the treatment of BCP-ALL; (vi) the treatment of T-ALL; or (vii) the treatment of lymphoblastic leukaemia;wherein the BCL2 inhibitor is administered in combination with a CREBBP and / or EP300 inhibitor.

[0092] In an embodiment, the BCL2 inhibitor is administered simultaneously, separately or sequentially with the CREBBP and / or EP300 inhibitor.

[0093] The present invention aslo provides the use of a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a CREBBP and / or EP300 inhibitor or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients, in the manufacture of a medicament for use in (i) the treatment of ALL; (ii) the treatment of B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia; (iii) the treatment of B-ALL, BCP-ALL or T-ALL; (iv) the treatment of B-ALL; (v) the treatment of BCP-ALL; (vi) the treatment of T-ALL; or (vii) the treatment of lymphoblastic leukaemia; wherein the CREBBP and / or EP300 inhibitor is administered in combination with a BCL2 inhibitor.

[0094] In an embodiment, the CREBBP and / or EP300 inhibitor is administered simultaneously, separately or sequentially with the BCL2 inhibitor.

[0095] The present invention also provides a method of treating: (i) ALL; (ii) B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia; (iii) B-ALL, BCP-ALL or T-ALL; (iv) B-ALL; (v) BCP-ALL; (vi) T-ALL; or (vii) lymphoblastic leukaemia. wherein the method comprises administering a therpetuically effective amount of a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a BCL2 inhibitor or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients, in combination with a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a CREBBP and / or EP300 inhibitor or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients .

[0096] In an embodiment, the BCL2 inhibitor is administered simultaneously, separately or sequentially with the CREBBP and / or EP300 inhibitor.

[0097] In a particular embodiment, BCL2 inhibitor is administered simultaneously with the CREBBP and / or EP300 inhibitor.

[0098] In a particular embodiment, BCL2 inhibitor is administered sequentially with the CREBBP and / or EP300 inhibitor.

[0099] In a particular embodiment, BCL2 inhibitor is administered separately with the CREBBP and / or EP300 inhibitor.

[0100] In a particular embodiment, a pre-treatment with the CREBBP and / or EP300 inhibitor is administered prior to commencing therapy with a BCL2 inhibitor.

[0101] The anti-proliferative effects of the combination therapy of the present invention has particular application in the treatment of human ALL cancers. This includes any cancer that has been unresponsive to a therapy comprising either a CREBBP and / or EP300 inhibitor or a BCL2 inhibitor alone.

[0102] In a particular embodiment, the combination therapy of the invention is suitable for the treatment of B-ALL.

[0103] The anti-cancer effect may arise through one or more mechanisms, including but not limited to, the promotion of an antitumour immune response, the regulation of cell proliferation, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures or within an organ), or the promotion of apoptosis (programmed cell death).

[0104] As indicated above, the BCL2 inhibitor may be any BCL2 inhibitor and the CREBBP and / or EP300 inhibitor may be any known CREBBP and / or EP300 inhibitor.

[0105] In one embodiment, the BCL2 inhibitor is venetoclax, or a pharmaceutically acceptable salt thereof; and the CREBBP and / or EP300 inhibitor is inobrodib or a pharmaceutically acceptable salt thereof. Pharmaceutical Compositions and Products

[0045] In another aspect, the present invention provides a pharmaceutical product comprising a combination of a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, and aCREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof for use in the treatment of ALL.

[0046] In one embodiment, the pharmaceutical product may comprise a kit of parts comprising separate formulations of a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, and a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof. The separate formulations of a BCL2 inhibitor and a CREBBP and / or EP300 inhibitor may be administered sequentially, separately and / or simultaneously.

[0047] In another embodiment, the pharmaceutical product is a kit of parts which comprises: a first container comprising a BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable adjuvant, diluent or carrier; and a second container comprising a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable adjuvant, diluent or carrier, and a container means for containing said first and second containers.

[0106] In one embodiment, the pharmaceutical product may comprise a one or more unit dosage forms (e.g. vials, tablets or capsules in a blister pack). In one embodiment, each unit dose comprises only one agent selected from the BCL2 inhibitor and the CREBBP and / or EP300 inhibitor. In another embodiment, the unit dosage form comprises both the a BCL2 inhibitor and the CREBBP and / or EP300 inhibitor, or pharmaceutically acceptable salts thereof.

[0107] In one embodiment the pharmaceutical product or kit of parts further comprises means for facilitating compliance with a dosage regimen, for instance instructions detailing how to administer the combination.

[0108] In one embodiment, the pharmaceutical product or kit of parts further comprises instructions indicating that the combination, as defined herein, can be used in the treatment of cancer.

[0109] In one embodiment, the pharmaceutical product is a pharmaceutical composition.

[0110] In one aspect, the present invention relates to a pharmaceutical composition comprising a combination of BCL2 inhibitor as defined herein, or a pharmaceutically acceptable salt thereof, and a CREBBP and / or EP300 inhibitor as defined herein, or apharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0111] The pharmaceutical compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing or as a suppository for rectal dosing).

[0112] The pharmaceutical compositions of the invention will typically be for parenteral or oral administration.

[0113] The pharmaceutical compositions may be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and / or preservative agents.

[0114] An effective amount of each component of the combination therapy will be present. Such an amount is an amount sufficient to treat or prevent a cancer condition referred to herein, slow its progression and / or reduce the symptoms associated with the condition.

[0115] The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the individual treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of active agent (more suitably from 0.5 to 100 mg, for example from 1 to 30 mg) compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.

[0116] The size of the dose for therapeutic or prophylactic purposes of a combination of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

[0117] In using a combination of the invention for therapeutic or prophylactic purposes it will generally be administered within a therapeutically effective dose of the particular agentconcerned. These dosages are known in the art and will vary from one agent to another. The dosage may, for example, be in the range of 0.1 mg / kg to 30 mg / kg body weight. The dosing schedule will also vary from one inhibitor to another. Suitable dosing schedules are known in the art. Routes of Administration

[0118] The combination of the invention or pharmaceutical compositions comprising said combination may be administered to a subject by any appropriate or convenient route of administration, whether systemically / peripherally or topically (i.e., at the site of desired action).

[0119] Depending on the nature of the agent, routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.

[0120] Suitably, the the agents of the present invention are administered by the oral, intravenous or intrathecal route. Combinations with additional therapeutic agents

[0121] The combination treatments defined herein may be applied as a sole therapy for the treatment of the specified condition or it may involve, in addition to the combination therapy of the present invention, one or more additional therapies (including treatment with another therapeutic agent, surgery or other therapeutic interventions such as radiotherapy in the oncology setting).

[0122] Typically, the other therapeutic agent used in combination with the combination therapy of the present invention will be one or more therapeutic agents used as the standard of care for the treatment of the disease or condition concerned. The other therapeutic agent may include, for example, another drug used for the treatment of the condition concerned, or an agent that modulates the biological response to the combination therapy of the invention, such as, for example, an immunomodulatory agent.

[0123] Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this invention within the dosage range described hereinbefore and the other pharmaceutically-active agent within its approved dosage range.

[0124] For example, the combination therapies defined hereinbefore may be applied as a sole therapy or may involve, in addition to the compound of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may further include one or more of the following categories of anti-tumour agents:- other antiproliferative / antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites (for example gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere and polokinase inhibitors); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); cytostatic agents such as antioestrogens (for example tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5 ^-reductase such as finasteride; anti-invasion agents [for example c-Src kinase family inhibitors like 4-(6-chloro-2,3- methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4- yloxyquinazoline (AZD0530; International Patent Application WO 01 / 94341), N-(2-chloro-6- methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole- 5-carboxamide (dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661) and bosutinib (SKI-606), and metalloproteinase inhibitors like marimastat, inhibitors of urokinase plasminogen activator receptor function or antibodies to Heparanase];inhibitors of growth factor function: for example such inhibitors include growth factor antibodies and growth factor receptor antibodies (for example the anti-erbB2 antibody trastuzumab [Herceptin™], the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab [Erbitux, C225] and any growth factor or growth factor receptor antibodies disclosed by Stern et al. (Critical reviews in oncology / haematology, 2005, Vol.54, pp11-29); such inhibitors also include tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl)-7- methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD1839), N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6- acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib); inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; inhibitors of the platelet-derived growth factor family such as imatinib and / or nilotinib (AMN107); inhibitors of serine / threonine kinases (for example Ras / Raf signalling inhibitors such as farnesyl transferase inhibitors, for example sorafenib (BAY 43-9006), tipifarnib (R115777) and lonafarnib (SCH66336)), inhibitors of cell signalling through MEK and / or AKT kinases, c-kit inhibitors, abl kinase inhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinase inhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors; aurora kinase inhibitors (for example AZD1152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclin dependent kinase inhibitors such as CDK2 and / or CDK4 inhibitors; antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, [for example the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin™) and for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib (ZD6474), vatalanib (PTK787), sunitinib (SU11248), axitinib (AG-013736), pazopanib (GW 786034) and 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1- ylpropoxy)quinazoline (AZD2171; Example 240 within WO 00 / 47212), compounds such as those disclosed in International Patent Applications WO97 / 22596, WO 97 / 30035, WO 97 / 32856 and WO 98 / 13354 and compounds that work by other mechanisms (for example linomide, inhibitors of integrin ^v ^3 function and angiostatin)]; vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99 / 02166, WO 00 / 40529, WO 00 / 41669, WO 01 / 92224, WO 02 / 04434 and WO 02 / 08213; an endothelin receptor antagonist, for example zibotentan (ZD4054) or atrasentan;antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense; gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies.

[0125] In a particular embodiment, the combination therapies defined hereinbefore may involve, in addition to the combination therapy of the invention, conventional surgery or radiotherapy or standard chemotherapy (e.g. treatment with systemic anticancer therapy, including cytotoxic chemotherapy, glucocorticoids, immunotherapies (e.g. monoclonal antibodies, bispecific antibodies, antibody drug conjugates, CAR-T cells etc.), tyrosine kinase inhibitors, bone marrow transplantation and radiotherapy).

[0126] Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the combination therapy of this invention within the dosage range described hereinbefore and the other pharmaceutically-active agent within its approved dosage range.

[0127] According to this aspect of the invention there is provided a combination as defined hereinbefore for use in the treatment of a cancer as defined herein, comprising a combination therapy of the invention as defined hereinbefore, and another anti-tumour agent.

[0128] According to this aspect of the invention there is provided a combination for use in the treatment of cancer as defined herein, comprising a combination therapy of the invention as defined hereinbefore, and any one of the anti-tumour agents listed herein above.

[0129] In a further aspect of the invention there is provided a combination product of the invention for use in the treatment of cancer in combination with another anti-tumour agent, optionally selected from one listed herein above. EXAMPLES PART A

[0130] Reference is made to the accompany figures, in which: Figure 1 shows CREBBP-mutated B-ALL shows increased sensitivity to Venetoclax: A, Schematic of genome editing strategy used to engineer 697WT(black) into isogenic CREBBP 697KI(blue) and 697KO(yellow) clones. B, Cell-based drug screening was performed using a panel of 32 small molecule compounds predicted to have differential sensitivity between CREBBP WT and mutant lines. 72h viability was measured by MTS assays using a wide concentration range between 3pM to 30µM in triplicate and repeated using narrower concentration ranges to define IC50where appropriate. Results are presented as an IC50ratio of 697KI(blue) and 697KO(yellow) clones compared to WT. C, Dose response curves of two CREBBP / EP300 inhibitors Inobrodib (left) and A485 (right) showing enhanced sensitivity of 697KI(blue) compared to 697WT(black) in 72h MTS viability assay. Performed in triplicate, mean ± SD. D, Dose response curve of 697WT(black) and 697KI(blue) lines to Venetoclax in 72h MTS viability assay. Performed in triplicate, mean ± SD. E, Growth curve of 697WT(black) and 697KI(blue) grown in the presence of either DMSO vehicle (dotted lines) or 20nM Venetoclax (solid lines). F, Mitochondrial depolarisation as assessed by staining for JC1 by flow cytometry (488nm 530 / 30) in response to DMSO vehicle, or Venetoclax at 20 or 2000nM in 697WT(black) and 697KI(blue) cell lines. G, Representative flow cytometry plots of externalization of Annexin V (reported by APC) in response to 24h exposure to DMSO vehicle (left) or Venetoclax 20nM (right) in 697WT(top) and 697KI(bottom) cell lines. Viability is assessed by 7AAD exclusion. H, Summary of 3 replicate experiments measuring proportion of viable 7AAD-veAnnexin-V+veearly apoptotic cells. Mean ± SD, 2 way ANOVA****, P <0.0001. Figure S1 shows CREBBP-mutated B-ALL shows increased sensitivity to Venetoclax:A, Summary of genome editing strategy. Top: Two single base substitutions were introduced by CRISPR directed homologous recombination to: i) generate the R1446C mutation; and ii) remove the protospacer adjacent motif (PAM) to prevent further Cas9 binding and repeat cutting of successful edits. Bottom Left: Results of amplicon sequencing showing a homozygous edit in 697KIcells and a compound heterozygous edit in two REH mutant clones. The alternative sequences for the remaining two alleles were sequenced on TOPO-TA cloned amplicon fragments (sequences, amino acid substitution and TOPO-TA clonal frequency are shown in table below). Bottom Right: Western blot of CREBBP vs Vinculin protein in 697 edited clones, confirming loss of protein in 697KOcells. B, Growth curves of 697WT(black), 697KI(blue) and 697KO(yellow) grown in the presence of DMSO vehicle (hashed lines) or 10nM Dexamethasone (solid lines). C, Dose response curves of two CREBBP / EP300 inhibitors A485 (top) and Inobrodib (bottom) showing enhanced sensitivity of 697KO(yellow) compared to 697WT(black) in 72h MTS viability assay. Performed in triplicate, mean ± SD. D, Dose response curve of 697WT(black) and 697KO(yellow) lines to Venetoclax in 72h MTS viability assay. Performed in triplicate, mean ± SD. E, Dose response curve of REHWT(green) and two isogenic CREBBP-mutant clones (purple and red) to Venetoclax in 72h MTS viability assays. Performed in triplicate, mean ± SD. F, Western blot for cleaved PARP (top) and cleavage of pro-caspase 3 (middle) in 697WT(left) and 697KI(right) cell lines. Cells were incubated with DMSO vehicle or Venetoclax at either 20nM or 2000nM concentrations. Actin is shown as a loading control (bottom). Figure 2 shows Venetoclax exerts its effect on CREBBP-mutated B-ALL by on-target inhibition of BCL2: A, Schematic of doxycycline-inducible shRNA KD system competitive co-culture assay.697WTand 697KIcells were stably transfected with two separate doxycycline- inducible shRNAs targeting BCL2, reported by mCherry, or a control shRNA targeting Xenopus Renilla, reported by GFP. BCL2 and Renilla shRNA-expressing cells were mixed in equal numbers, doxycycline added to the media (500ng / ml) to induce shRNA expression and the proportion of cells surviving BCL2 / Renilla-KD analysed by daily flow cytometry. Created with BioRender.com.B, BCL2 shRNA KD competitive proliferation assay for two different BCL2-targeting shRNAs are presented, showing the ratio of BCL2-targeting shRNA (mCherry) vs. Renilla control (GFP), in 697KInormalized to 697WTas a percentage. Performed in triplicate, mean ± SD. C, Representative flow cytometry plots of externalisation of Annexin V (APC) in 697WT(top) and 697KI(bottom) cell lines in response to doxycycline-induced shRNAs targeting Renilla (left), or two different BCL2-targeting shRNAs (middle and right). Day 6 post induction. Viability is assessed by 7AAD exclusion. D, Summary of replicate experiments measuring proportion of viable 7AAD-veAnnexin V+veearly apoptotic cells normalized to doxycycline-induced Renilla control. Analysed 6 days after induction, performed in triplicate, mean ± SD, 2 way ANOVA****, P <0.0001. E, Dose response curve of 697WT(black) and 697KI(blue) to Navitoclax in 72h MTS viability assays. Performed in triplicate, mean ± SD. Chemical structure of Navitoclax is shown below. Images from Selleckchem. F, Dose response curve of 697WT(black) and 697KI(blue) to A1155463 (left) and AZD5991 (right) in 72h MTS viability assays. Performed in triplicate, mean ± SD. Chemical structure of drugs are shown below. Images from Selleckchem. Figure S2 shows Venetoclax exerts its effect on CREBBP-mutated B-ALL by on-target inhibition of BCL2: A, Schematic of doxycycline-inducible shRNA KD system linked to fluorescent reporter proteins (25). Created with BioRender.com. B, Doxycycline-induced KD of two different BCL2-targeting shRNAs measured by RT- qPCR. Triplicate measurements, internally normalised to GAPDH and presented as a ratio to Renilla control. Day 3 post induction. Mean ± SEM. C, Western blot of BCL2 KD by two different doxycycline-inducible shRNAs in 697WT(left) and 697KI(right) cells. Day 3 post induction. Beta-Tubulin is presented as a loading control. D, Secondary antibody fluorescence intensity from Fig. S2C normalized to Beta- Tubulin loading control. E, Representative flow cytometry histograms of Annexin V (APC) externalization in response to escalating doses of Navitoclax in 697WT(left) and 697KI(right).F, Summary of replicate experiments measuring Annexin V+vecells normalized to DMSO-treated vehicle control cells. Performed in triplicate, mean ± SD, 2 way ANOVA ****, P <0.0001. Figure 3 shows CREBBP-mutated B-ALL shows significant cell cycle and metabolic dysregulation: A, Summary of most significantly down-regulated (left) and up-regulated (right) KEGG (Kyoto Encyclopaedia of Gene and Genomes) pathways from DEGs of RNAseq analysis comparing DMSO vehicle-treated 697KIwith 697WT. B, GSEA analysis of ranked genes of RNAseq analysis comparing DMSO vehicle- treated 697KIwith 697WT. NES: normalized enrichment score; FDR false discovery rate (52). C, Comparison of BCL2 expression levels in 697WT(black) and 697KI(blue) cells by RNAseq. Each dot represents one sample. Bars show mean fragments per kilobase of transcript per million fragments mapped (FPKM) value ± SD, n=3, significance calculated by unpaired t test **, P=0.0038. D, Comparison of CDKN2A expression levels in 697WT(black) and 697KI(blue) cells by RNAseq. Each dot represents one sample. Bars show mean FPKM value ± SD, n=3, significance calculated by unpaired t test, P=0.0002. E, Proliferation of untreated 697WT(black), 697KI(blue) and 697KO(Yellow) cells measured by direct counting. Performed in triplicate, mean ± SD. F, Glycolytic rate measured by extracellular acidification rate (ECAR) using Seahorse (Agilent) Glycostress test in 697WT(black) and 697KI(blue) cells. Mean ± SEM. Left panel: Representative ECAR plot over time. Right panel: Summary of maximal ECAR. Each dot represents a single replicate acquired from two separate experiments. Significance calculated by unpaired t test****, P <0.0001. G, Mitochondrial oxygen consumption rate (OCR) measured using Seahorse (Agilent) Mitostress test. Left panel: Representative OCR plot over time. Mean ± SEM. Right panel: Summary of basal OCR (left), maximal OCR (middle) and spare respiratory capacity (right). Each dot represents a single replicate acquired from two separate experiments. Significance calculated by unpaired t test****, P <0.0001. Figure S3 shows CREBBP-mutated B-ALL shows significant cell cycle and metabolic dysregulation:A, Number of up- and down-regulated DEGs (P and FDR <0.05) by RNAseq comparing 697KIwith 697WTDMSO vehicle-treated cells. B, GSEA of ranked RNAseq expression of 697KIversus 697WTDMSO vehicle-treated cells for known Crebbp target genes in CrebbpKOmouse germinal centre lymphocytes (26). NES: normalized enrichment score; FDR false discovery rate. C, KEGG pathway showing differential expression of apoptotic regulators from RNAseq comparing 697KIversus 697WTDMSO vehicle-treated cells. D, GSEA of ranked RNAseq expression of 697KIversus 697WTDMSO vehicle-treated cells. NES: normalized enrichment score; FDR false discovery rate. E, Significant up-regulated pathways identified by Gene Ontology (GO) database analysis of RNAseq comparing 697KIversus 697WTDMSO vehicle-treated cells. Figure 4 shows Venetoclax induces ferroptotic cell death in CREBBP-mutated B-ALL: A, Summary of most significantly down-regulated (top) and up-regulated (bottom) KEGG pathways from DEGs of RNAseq analysis comparing Venetoclax-treated 697KIwith 697WT. B, GSEA analysis of ranked genes of RNAseq analysis comparing Venetoclax-treated 697KIwith 697WT. NES: normalized enrichment score; FDR: false discovery rate (53, 54). C, Cellular viability assessed by flow cytometry of 697WT(black) and 697KI(blue) cells treated with increasing doses of Venetoclax at 24h either with exposure to the cell- permeant pan-caspase inhibitor Z-VAD (hashed) or DMSO control (plain). Performed in triplicate, mean ± SD, analysed by 2 way ANOVA,****, P <0.0001. D, Cellular viability assessed by flow cytometry of 697WT(black) and 697KI(blue) cells treated with increasing doses of Venetoclax at 24h, either with exposure to the ferroptotic inhibitor Liproxstatin 1 (hashed) or DMSO control (plain). Performed in triplicate, mean ± SD, analysed by 2 way ANOVA,****, P <0.0001. E, Lipid peroxidation in Venetoclax-treated 697WT(black), 697KI(blue) and 697KO(yellow) cells assessed by BODIPYC11 expressed as a ratio of FL1:FL3 (488nm 530 / 30:610 / 20) mean fluorescence intensity (MFI) normalized to untreated cells. Performed in triplicate, each dot represents a single sample, mean ± SD, two way ANOVA,****, P <0.0001. F, Summary of basal (left) and maximal (right) mitochondrial oxygen consumption rate (OCR) measured using Seahorse (Agilent) Mitostress test in 697WT(black), 697KI(blue)cells exposed to increasing concentrations of Venetoclax. Each dot represents a single replicate acquired from two separate experiments. One way ANOVA****, P <0.0001. G, Viability of 697WT(left column for each erastin concentration, black) and 697KI(right column for each erastin concentration, blue) cells following 48h exposure to Erastin at 15 or 30µM. Viability assessed by 7AAD exclusion usingflow cytometry. Mean ± SD of three experiments performed in triplicate. Two way ANOVA; *, P <0.014. H, Lipid peroxidation in in RSL3-treated 697WT(left column for each RSL3 dose, black) and 697KI(right column for each RSL3 dose, blue) cells assessed by BODIPYC11expressed as a ratio of FL1:FL3 (488nm 530 / 30:610 / 20) meanfluorescence intensity (MFI) normalized to untreated cells. Mean ± SD of 2 experiments performed in triplicate. Two way ANOVA, ***, P=0.0001. Figure S4 shows Venetoclax induces ferroptotic cell death in CREBBP-mutated B-ALL: A, Four-way interaction model identifies 1487 genes (FDR 0.05) differentially expressed specifically in Venetoclax-treated 697KIcells. B, Representative histogram of flow cytometric BODIPYC11staining (488nm 530 / 30) of 697WT(left), 697KI(Middle) and 697KO(right) cells in response to increasing doses of Venetoclax (DMSO: Blue, Venetoclax 20nM: Red, Venetoclax 200nM: Orange). C, Left panel: Representative histogram of flow cytometric BODIPYC11 staining (488nm 530 / 30) of 697WT(left) and 697KI(right) cells in response to 200nM Navitoclax (red) or DMSO vehicle (blue). Right panel: Summary BODIPYC11 staining 697WT(black) and 697KI(blue) cells in response to 50nM Navitoclax, 200nM Navitoclax or DMSO vehicle normalized to DMSO vehicle. Performed in triplicate, each dot represents a single sample, mean ± SD, two way ANOVA, P = 0.0033. D, Left panel: Representative histogram of flow cytometric BODIPYC11 staining (488nm 530 / 30) of REHWT(left) and REHMut1(right) cells in response to 200nM of Venetoclax. Right panel: Summary BODIPYC11 staining REHWT(green) and REHMut1(purple) cells in response to Venetoclax normalized to DMSO vehicle. Performed in triplicate, each dot represents a single sample, mean ± SD, two way ANOVA,****, P <0.0001. Figure 5 shows pharmacological inhibition of CREBBP function can sensitise B-ALL to Venetoclax in-vitro:A, Dose response curve of 697WT(black) and 697WTpre-treated with 3 days of A485 (pale blue) to Venetoclax in 72h MTS viability assays. Performed in triplicate, mean ± SD. B, Three-dimensional diffusion plot of ZIP synergy score to combined doses of synchronous A485 and Venetoclax (peak ZIP 62.24) in 697WTcells. Viability measured by 72h MTS assay. C, Summary BODIPYC11staining 697WT(black) and 697WTtreated with A485 (blue) cells in response to Venetoclax. FL1:FL3 signal normalized to DMSO vehicle. Performed in triplicate, each dot represents a single sample, mean ± SD, two way ANOVA,***, P <0.0006,****, P <0.0001. D, Summary of basal (left) and maximal (middle) mitochondrial oxygen consumption rate (OCR) and spare respiratory capacity (right) measured using Seahorse (Agilent) Mitostress test in 697WTcells treated with A485 (pale blue) or DMSO vehicle (black). Each dot represents a single replicate acquired from two separate experiments. Unpaired t test****, P <0.0001. E, Growth curve of 697WTcells treated with Venetoclax 100nM and / or A485 at two different doses (100nM and 300nM). Duplicate measurements, mean ± SD presented as log2 accumulative fold change. F, Annexin V (APC) externalization assessed by flow cytometry in 697WTcells treated with Venetoclax 100nM and / or A485 at two different doses (100nM and 300nM). Duplicate measurements, each dot represents a single replicate, mean fluorescent intensity (MFI) ± SD normalized to DMSO control. G, Dose response curve of NALM6WT(solid line) and NALM6WTpre-treated with 3 days of A485 (hashed line) to Venetoclax in 72h MTS viability assays. Performed in triplicate, mean ± SD. H, Summary BODIPYC11 staining NALM6WT(red) and NALM6WTtreated with A485 (brown) cells in response to Venetoclax. FL1:FL3 signal normalized to DMSO vehicle. Performed in triplicate, each dot represents a single sample, mean ± SD, two way ANOVA,****, P <0.0001. I, Three-dimensional diffusion plot of ZIP synergy score to combined doses of synchronous A485 and Venetoclax. Viability measured by 72h MTS assays in KOPN8 (MLL::ENL) (peak ZIP 12.66), RS4-11 (MLL::AF4) (peak ZIP 27.08) and SUPB15 (BCR::ABL1) (peak ZIP 13.86) Venetoclax-sensitive cell lines.Figure S5 shows pharmacological inhibition of CREBBP function can sensitise B-ALL to Venetoclax in-vitro: A, Dose response curve of REHWT(solid line) and REHWTpre-treated with 3 days of A485 (hashed line) to Venetoclax in 72 hour MTS viability assays. Performed in triplicate, mean ± SD. B, Representative histogram of flow cytometric BODIPYC11staining of 697WT(top) and 697WTpre-treated with A485 (bottom) in response to increasing doses of Venetoclax. C, Dose response curve of 697WT(black) and 697WTpre-treated with 3 days of Inobrodib (pale blue) to Venetoclax in 72h MTS viability assays. Performed in triplicate, mean ± SD. D, Three-dimensional diffusion plot of ZIP synergy score to combined doses of synchronous Inobrodib and Venetoclax (peak ZIP 37.58). Viability measured by 72h MTS assay. E, Representative mitochondrial oxygen consumption rate (OCR) measured using Seahorse (Agilent) Mitostress test in 697WT(solid line) compared to 697WTtreated with A485 (hashed line). Mean ± SEM. F, Representative mitochondrial oxygen consumption rate (OCR) measured using Seahorse (Agilent) Mitostress test in REHWT(solid line) compared to REHWTtreated with A485 (hashed line). Mean ± SEM. G, Summary of basal (left) and maximal (middle) mitochondrial oxygen consumption rate (OCR) and spare respiratory capacity (right) measured using Seahorse (Agilent) Mitostress test in REHWTcells treated with A485 or DMSO vehicle. Each dot represents a single replicate acquired from two separate experiments. Unpaired t test****, P <0.0001. H, Representative histogram of flow cytometric BODIPYC11 staining of NALM6WT(left) and NALM6WTpre-treated with A485 (right) in response to Venetoclax. Figure 6 shows genetic or pharmacological inhibition of CREBBP sensitises B-ALL to Venetoclax in-vivo: A, Schema of 697WTvs. 697KIin vivo drug dosing protocol. NSG mice were intravenously injected with 697WTvs.697KIluciferized cell lines. At 8 days post injection animals were administered Venetoclax (100mg / kg) or vehicle control by oral gavage. Mice were imaged by BLI as indicated. N=6 per group.B, Representative BLI images of mice engrafted withcells at days 11 and 17 of treatment. C, Average BLI radiance of mice engrafted with 697WTcells at days 11 and 17 of treatment (log p / s / cm2 / sr). Mean ± SEM. D, Representative BLI images of mice engrafted with 698KIcells at days 11, 17 and 21 of treatment. E, Average BLI radiance of mice engrafted with 697KIcells at days 11, 17 and 21 of treatment (log p / s / cm2 / sr). Mean ± SEM. F, Overall survival of NSG mice engrafted with 697WTvs.697KIB-ALL cells treated with oral Venetoclax (100mg / kg) or vehicle control. 30 day maximal treatment window is indicated. N=6 per group. Significance calculated by Mantel Cox log-rank test. G, Overall survival of NSG mice engrafted with 697WTB-ALL cells treated with oral Inobrodib (20mg / kg) or a combination of Inobrodib (20mg / kg) plus Venetoclax (100mg / kg) (n=4 per group). Engraftment control groups were treated with Venetoclax only (n=2) or vehicle control (n=1). Treatment began at day 8 post-transplant. Significance calculated by Mantel Cox log-rank test. Figure S6 shows genetic or pharmacological inhibition of CREBBP sensitises B-ALL to Venetoclax in-vivo: A, In vitro BLI measurements of a serial dilution of luciferase-expressing 697WTvs. 697KIB-ALL cells (log p / s / cm2 / sr). B, Baseline weights of NSG mice treated in Fig.6A-F. Mean ± SEM. C, Average BLI radiance of mice engrafted with 697WTor 697KIcells treated with Venetoclax or vehicle control at days 11, 17 and 21 and 37 (697KIonly) of treatment (log p / s / cm2 / sr). Mean ± SEM. D, Average BLI radiance of mice engrafted with 697WTor 697KIcells treated with Venetoclax or vehicle control at days 11, 17 and 21 (697KIonly) of treatment (log p / s / cm2 / sr) normalized to untreated 697KIrecipients. Mean ± SEM. E, Baseline weights of NSG mice treated in Fig.6G. Bar represents median average. Figure 7 shows a dose response curve of 697WT(black) and 697KI(blue) to the BCL2 inhibitor Sonrotoclax in 72h MTS viability assays. Performed in triplicate, mean ± SD.Figure 8A shows the viability of 697WT(left two columns for each erastin concentration, black) and 697KI(right two columns for each erastin concentration, blue) cells comparing parental (plain) and resistant (hashed) lines following 48h exposure to Erastin at 15 or 30µM. Viability assessed by 7AAD exclusion usingflow cytometry. N=3, mean ± SD. Two way ANOVA, ****, P <0.0001. Figure 8B shows a three-dimensional diffusion plot of ZIP synergy score to combined doses of synchronous Erastin and Venetoclax in 697WT WT-Res(left) (peak ZIP 41.37), 697 (middle) (peak ZIP 33.34) and 697KI-Res(right) (peak ZIP 22.69). Viability measured by 72h MTS assay. Results CREBBP-mutated B-ALL shows increased sensitivity to Venetoclax

[0131] To identify candidate therapeutics that can specifically target CREBBP-mutated high- risk B-ALL, a synthetic-lethal drug screen in human B-ALL cell lines was undertaken. The CREBBP wild-type (WT) B-ALL cell line 697 (driven by the recurrent E2A::PBX1 fusion) was genome-engineered by CRISPR-Cas9 homologous recombination to introduce a recurrent “hotspot” mutation at arginine 1446 (CREBBPR1446C), which has been implicated as exerting a dominant-negative effect on CREBBP acetyltransferase activity (5). A number of clones were generated, including a homozygous CREBBPR1446Cknock-in mutant clone (hereafter, 697KI) and a mutant clone containing two frameshift mutations resulting in a complete knockout of CREBBP protein (hereafter, 697KO) (Fig. 1A; Fig. S1A). In addition, for use in validation studies, the ETV6::RUNX1-driven cell line REH (which has three WT copies of CREBBP) was also edited, resulting in two compound-heterozygous mutated clones, each including a single allele of the CREBBPR1446CHAT mutation, alongside presumed deleterious mutations of the other two alleles (Fig. S1A).

[0132] The 697 isogenic cell lines were subjected to a targeted drug screen, using a wide range of concentrations, focussed on clinically-actionable drugs in classes that have either been implicated or hypothesized to show differential sensitivity patterns in published models of B cell lymphoma and other CREBBP-mutated malignancies (4, 5, 11-13, 18, 20, 21). CREBBP-mutated 697 cells were not differentially sensitive to traditional cytotoxic chemotherapy, and paradoxically showed a degree of sensitisation to the glucocorticoid dexamethasone, used in current ALL induction regimens (Fig.1B and Fig. S1B) (5, 12, 13).

[0133] As anticipated, and validating the screen design, inhibitors of the CREBBP paralogue EP300 (the CREBBP / EP300-specific bromodomain inhibitor Inobrodib and theCREBBP / EP300 acetylase inhibitor A485) exhibited synthetic lethality, consistent with previous reports in B-cell lymphoma (Fig.1B and C and Fig. S1C) (18).

[0134] However, unexpectedly, the most potent hit identified from the screen was the clinical-grade BCL2 inhibitor Venetoclax, which showed a 2-log10-fold reduction in IC50, in both 697KIand 697KOclones (Fig. 1B and D and Fig.S1D). These findings were validated in the isogenic REH lines, with a 1-log10-fold reduction in IC50demonstrated in both mutant clones (Fig. S1E). This sensitization to Venetoclax was confirmed in in-vitro proliferation assays by direct cell counting (Fig.1E). Upon low-dose (20nM) Venetoclax exposure, CREBBP-mutated 697 cells showed enhanced evidence of markers of programmed cell death, including mitochondrial depolarisation, externalisation of Annexin V and induction of cleaved PARP and caspase-3 (Fig 1F-H; Fig. S1F), consistent with the known mechanism of action of Venetoclax through the induction of apoptotic programmed cell death.

[0135] Overall, this focussed drug screen demonstrates that CREBBP-mutated B-ALL is: i) not generically chemo-resistant, and ii) identifies a number of clinically-actionable agents for use in CREBBP-mutated high-risk B-ALL, including dexamethasone, EP300 inhibitors and a potent sensitization to the BCL2 inhibitor Venetoclax. Venetoclax exerts its effect on CREBBP-mutated B-ALL by on-target inhibition of BCL2

[0136] The mechanism of action of Venetoclax was investigated in more detail. Venetoclax was developed to induce apoptosis through direct inhibition of BCL2 binding to the key pro- cell death proteins BAK and BAX. However, more recently, it has been shown to have alternative mechanisms of action, in particular on metabolism and self-renewal, including potential BCL2-independent effects (22-24). To test whether the mechanism of action of Venetoclax in CREBBP-mutated B-ALL was mediated through on-target BCL2 inhibition, a doxycycline-inducible shRNA knock-down (KD) system was employed, where shRNA expression was directly linked to a fluorescent reporter (Fig. S2A-D) (25).

[0137] Co-culture of 697WTor 697KIcells expressing one of two unique BCL2-targeting shRNAs (reported by mCherry) competed against cells expressing an shRNA targeting the negative-control Xenopus Renilla gene (reported by green fluorescent protein (GFP)) showed that 697KIcells exhibited a marked competitive disadvantage upon BCL2-KD, when compared to 697WT(Fig. 2A & 2B). This was associated with significant externalization of Annexin V, consistent with the induction of programmed cell death (Fig 2C & D).

[0138] Furthermore, 697KIcells also showed differential sensitivity to the structurally- unrelated clinical-grade dual BCL2 / BCLXL inhibitor Navitoclax (Fig 2E; Fig. S2E & F).Conversely, specific inhibitors of other antiapoptotic proteins (BCLXL and MCL1) showed no specificity for CREBBP-mutated cells (Fig 2F), confirming this to be a BCL2-specific effect.

[0139] Collectively these studies demonstrate that Venetoclax induces programmed cell death in CREBBP-mutated B-ALL by on-target BCL2 inhibition and that the sensitivity of 697KIcells is specific to BCL2 and does not occur through other anti-apoptotic proteins. CREBBP-mutated B-ALL shows significant cell cycle and metabolic dysregulation

[0140] To further explore the mechanism of action of Venetoclax in CREBBP-mutated 697 cells, the present inventors undertook bulk RNA sequencing of 697WTand 697KIcells, after 24 hours exposure to either dimethyl sulfoxide (DMSO) vehicle, or low-dose Venetoclax (20nM – the IC50of 697KIcells). Consistent with the role of CREBBP as a transcriptional co-activator, the majority of differentially expressed genes (DEGs) between the DMSO-vehicle treated 697WTand 697KIcells were down-regulated (Fig. S3A). Furthermore, Gene Set Enrichment Analysis (GSEA) showed a marked down-regulation of published CREBBP target genes from a mouse lymphoma model (NES -1.67, FDR 0.00) supporting the role of the CREBBPR1446Cmutation as inhibitory to CREBBP function as a transcriptional co-activator (Fig. S3B) (26).

[0141] GSEA analysis also showed significant down-regulation of signatures associated with apoptosis, in consonance with the functional experiments above (Fig.1F-H, Fig.3A and B). However, gene-specific examination of differential expression of apoptotic regulators by KEGG pathway analysis showed a mixed picture, affecting the expression of both pro- and anti-apoptotic genes, including a small but significant up-regulation of BCL2 itself (P=0.0038) (Fig 3C; Fig. S3C).

[0142] More broadly, KEGG and GSEA pathway analyses showed a differential down- regulation of cell cycle and signalling pathways in 697KI(Fig 3A and B; Fig. S3D). Downregulation of cell cycle-associated transcriptional signatures was associated with a marked up-regulation of the tumour suppressor CDKN2A (encoding the negative regulator of cell cycle P16INK4a-ARF), which is commonly mutated in B-ALL (Fig.3D). In keeping with this, it was confirmed that a relative reduction in proliferative capacity in both 697KIand 697KOcells compared to 697WTby proliferation assays (Fig.3E). This was associated with a significantly increased proportion of 697KIcells in G1 phase alongside reduced Early S / G2-S-M phases assayed by the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) GEMININ-CDT1 cell cycle reporter system confirming a significant defect in cell cycle progression. The majority of transcriptionally up-regulated KEGG and Gene Ontology (GO) pathways were indicative of metabolic dysfunction including and GSEA showed dysregulation of fatty acid oxidation and hypoxic gene signatures (Fig. 3A and B). Given the close association of cell cycle andmetabolism, coupled with the known role of both BCL2 and Venetoclax in disturbing mitochondrial respiration (24), the baseline metabolic differences between 697WTand 697KIcells using in-vitro Seahorse (Agilent) assays of both glycolysis and oxidative phosphorylation (OxPhos) were analysed. Unexpectedly, 697KIcells consistently showed increased rates of both glycolysis and OxPhos, including higher rates of both basal and maximal respiration, and an increase in spare respiratory capacity (Fig.3F and 3G).

[0143] Collectively these findings suggest that loss of CREBBP acetyltransferase function in the 697 cell line described herein model results in significant transcriptional dysregulation, affecting multiple cellular processes including apoptosis, cell cycle and metabolism. Venetoclax induces ferroptotic cell death in CREBBP-mutated B-ALL

[0144] To explore the transcriptional impact of low-dose Venetoclax treatment of 697KIcells, a four-way interaction model to identify genes specifically dysregulated in the 697KIcells upon Venetoclax treatment was employed (Fig. S4A). KEGG pathway analyses of these gene sets showed further down-regulation of cell cycle-associated genes and enrichment for metabolic pathways and ferroptosis when 697KIcells were treated with Venetoclax (Fig. 4A). Furthermore, GSEA showed marked up-regulation of genes associated with multiple metabolic processes, ROS scavenging, ferroptosis and the unfolded protein response (Fig. 4B).

[0145] Ferroptosis is a distinct form of programmed cell death resulting from iron-catalyzed reactive oxygen species (ROS)-mediated damage to unsaturated membrane lipids (27). It commonly co-associates with apoptosis and has been associated with expression of canonical programmed cell death markers, including Annexin V externalization (28). Evidence of ferroptosis upon Venetoclax treatment specifically occurring in 697KIcells using in-vitro assays has been demonstrated. Exposure to the cell-permeable pan-caspase inhibitor Z-VAD partially rescued cell viability to high-dose Venetoclax (2000nM) in both 697WTand 697KIcells, indicating a role for intrinsic, caspase-mediated apoptotic cell death induced by high Venetoclax doses in both cell lines (Fig.4C). Conversely, exposure to Liproxstatin 1, a specific inhibitor of ferroptosis-associated lipid peroxidation, rescued cell viability only in 697KIcells exposed to either low (20nM) or high-dose (2000nM) Venetoclax (Fig.4D), having no similar effect on 697WTcells. BCL2 inhibition with either Venetoclax or Navitoclax was also associated with evidence of enhanced lipid peroxidation in 697KI / KOand REHMutcells, as shown by BODIPYC11 staining, an indicator of lipid peroxidation (Fig.4E; Fig. S4B-D).

[0146] The intrinsic susceptibility of 697WTand 697KIcells to ferroptosis using direct inducers of Reduction-Oxidation (redox) stress was tested. 697KIcells were slightly moresusceptible to induction of ferroptosis by Erastin, which induces ferroptosis in RAS-mutated tumours, by both inhibiting system xc- (a cystine / glutamate antiporter that provides cysteine for the production of glutathione) and the VDAC family of mitochondrial outer membrane anion channels (Figure 4G). Similarly, inhibition of glutathione phospholipid peroxidase activity using the GPX4 inhibitor RSL3 induced significantly higher levels of lipid peroxidation by BODIPYC11staining in 697KIcells, further demonstrating their enhanced sensitivity to redox stress (Figure 4H). Metabolically, low dose (20nM) Venetoclax specifically resulted in a reduction in both basal and maximal OxPhos in 697KIcells, whereas only high-dose Venetoclax (2000nM) resulted in a marked reduction in OxPhos in both 697KIand 697WTcells, consistent with BCL2’s role in regulating mitochondrial outer membrane permeabilization (Fig. 4F).

[0147] Overall, these findings demonstrate that the major driver underlying the sensitivity of CREBBP-mutated cells to BCL2 inhibition is programmed cell death by ferroptosis, associated with underlying metabolic dysregulation and an increased dependence on ROS-scavenging pathways. Pharmacological inhibition of CREBBP function can sensitise B-ALL to Venetoclax in-vitro

[0148] The majority of B-ALL patients do not have CREBBP-mutations. Given the strength of the association with Venetoclax sensitization, most clearly demonstrated in the homozygous CREBBPR1446CHAT domain mutated 697KIcell line, and the unmet medical need in ALL therapy, it was hypothesized that pharmacological inhibition of CREBBPWTfunction could sensitise CREBBPWTB-ALL to BCL2 inhibitors.

[0149] Pre-treatment of 697WTor REHWTcells with the preclinical CREBBP / EP300 HAT inhibitor A485 almost perfectly phenocopied the degree of Venetoclax sensitization seen in the isogenic lines (Fig.5A; Fig. S5A). Furthermore, co-treatment with escalating doses of A485 and Venetoclax showed evidence of pharmacological synergy by ZIP score (Fig.5B). Further corroborating the isogenic findings, A485-sensitized Venetoclax cytotoxicity was associated with enhanced lipid peroxidation consistent with the induction of ferroptosis (Fig.5C; Fig. S5B). Similar results were also seen with the CREBBP / EP300-specific bromodomain inhibitor Inobrodib, which is currently undergoing early phase clinical trials in other hematological malignancies (Fig. S5C and Fig. S5D). In both cells, inhibition ofCREBBP / EP300 HAT activity by 300nM A485 was in basal and maximal OxPhos with an increase in spare respiratory capacity, similar to that seen in 697KIcells (Fig.5D; Fig. S5E-G). Exposure of 697WTcells to single agent A485 at 100 and 300nM did not result in either a significant loss of proliferation, nor evidence of programmed cell deathby annexin V externalisation, strongly suggesting that A485 treatment works by sensitising cells to Venetoclax-induced cytotoxicity (Fig.5E and Fig.5F).

[0150] These findings were extended into a panel of other human B-ALL cell lines. A485 sensitized the highly Venetoclax-resistant, BAX-mutated cell line NALM6 (IGH::DUX4 genotype) to Venetoclax treatment (Fig.5G). This effect was associated with increased lipid peroxidation by BODIPYC11staining, indicating a role for ferroptotic programmed cell death (Fig. 5H; Fig. S5H). Co-treatment with Venetoclax and A485 also showed evidence of pharmacological synergy in 3 highly Venetoclax-sensitive B-ALL lines driven by high-risk genetic drivers (BCR::ABL, KMT2A-rearranged), indicating that this interaction is conserved across diverse genetic subtypes of B-ALL (Fig.5I).

[0151] Collectively, these findings pharmacologically validate the findings of BCL2 sensitization identified in the CREBBP mutated cell lines and support a novel drug combination for clinical translation in B-ALL more broadly. Genetic or pharmacological inhibition of CREBBP sensitises B-ALL to Venetoclax in-vivo

[0152] It was tested whether Venetoclax could target CREBBP-mutated B-ALL in-vivo, where cell extrinsic factors and pharmacodynamic effects can result in reduced efficacy or highlight dose-limiting toxicities.

[0153] 697WTand 697KIcell lines were lentivirally-transfected with luciferase to allow disease response monitoring by in-vivo bioluminescent imaging (BLI), fluorescence activated cell sort purified to ensure comparable luciferase activity and engrafted into weight-matched, sub- lethally irradiated NOD-SCID-Gamma (NSG) mice (Fig. S6A and S6B). Upon confirmation of engraftment by BLI, mice were treated with daily oral Venetoclax (100mg / kg by oral gavage) for up to 30 days (Fig.6A). As anticipated, Venetoclax exposure was associated with limited disease control in 697WTcells as assessed by BLI progression (Fig.6B and C) and resulted in a small, but significant, prolongation of post-transplant overall survival (OS), with all Venetoclax 697WTrecipient animals succumbing within 15 days of starting treatment (median OS 19 vs. 22 days, p=0.0011) (Fig. 6F). 697KIrecipient mice treated with vehicle control succumbed to disease slightly later thanpotentially reflecting the defect in proliferation capacity characterized in-vitro (median OS 25 vs.19 days, p=0.0004) (Fig.6F); nevertheless, all 697KImice treated with vehicle succumbed by day 19 of treatment. Mice engrafted with 697KIcells and treated with Venetoclax demonstrated markedly improved disease control by BLI (Fig.6D & E; Fig. S6C and S6D). Only one Venetoclax-treated 697KIrecipient succumbed to disease within the treatment window, with the remaining 5 recipients gaining up to 2 weeksof survival after cessation of Venetoclax exposure (median OS 47.5 vs.25.5 days, p=0.0006) (Fig.6F).

[0154] It was also tested whether co-administration of the orally-available, early-phase CREBBP bromodomain inhibitor Inobrodib could sensitise 697WTcells to Venetoclax in-vivo (Fig. S6E). Once-daily oral dosing of Inobrodib (20mg / kg) only did not confer survival benefit compared to recipients treated with either vehicle or single agent Venetoclax, whereas recipient mice treated with both agents demonstrated a significant survival advantage (median OS combination 34.5 vs. Inobrodib only 24.5 days, p=0.0084) (Fig.6G).

[0155] Overall, these findings demonstrate that: i) oral Venetoclax can be highly efficacious in controlling CREBBP-mutated B-ALL in a preclinical in-vivo model; and ii) pharmacological inhibition of CREBBP is a tolerable and efficacious means of sensitizing B-ALL to Venetoclax in-vivo. Discussion

[0156] There is a pressing clinical need to develop novel treatment options for patients with high-risk B-ALL, such as that driven by LOF mutations in CREBBP. The present inventors have developed genome-engineered human B-ALL cell lines to test for differential patterns of drug resistance and sensitivity upon CREBBP loss. Using a highly-curated panel of clinically- tractable small molecules, it has been demonstrated that CREBBP mutation sensitizes cells to dexamethasone, inhibitors of residual CREBBP / EP300 function and most potently to the BCL2 inhibitor Venetoclax. Loss of CREBBP is associated with underlying defects in cell-cycle and metabolism and, upon BCL2 inhibition, CREBBP-mutated cells undergo loss of viability dominated by ferroptotic rather than apoptotic cell death. Pharmacological inhibition of CREBBP function synergises with BCL2 inhibition in multiple B-ALL cell lines, regardless of their genotype, validating the genetic findings and providing a readily translatable potential novel drug combination for B-ALL more broadly.

[0157] The model system described herein shows a number of significant similarities and differences to previously published work. It is noted that the global transcriptional profile of the CREBBP-mutated cells strongly overlaps with those published from a murine model of Crebbp- / -germinal centre B cells (26). It is also noted that other models of CREBBP-mutated B-ALL and B-cell lymphoma have implicated changes in cell cycle, metabolism, DNA damage response and apoptosis (4, 11, 20). Further corroborating the findings described herein, the present inventors also note that hypodiploid B-ALL, a genetic subtype that is highly associated with CREBBP mutations, has been specifically associated with Venetoclax sensitivity (21). Moreover, preliminary results from an early clinical phase trial of Venetoclax in relapsedpediatric malignancies (NCT03236857) have specifically highlighted responses in the small number of CREBBP-mutated B-ALL patients enrolled, providing further supportive evidence that the results will be clinically translatable (35).

[0158] The CREBBP-mutated cell lines did not show significant differential sensitivity to cytotoxic chemotherapy in the ex vivo setting. Previous reports have shown variable association of CREBBP LOF with chemoresistance in-vitro (11, 13, 20). The findings may be a feature specific to the model system described herein, or relate to the broad range of drug doses used in the screen, however, the cell lines also showed a small but significant sensitization to dexamethasone, suggesting that CREBBP LOF does not significantly alter responses to glucocorticoids (5, 12, 13). Overall, the findings described herein add to a growing body of evidence that CREBBP LOF does not in itself provoke significant cell-intrinsic chemoresistance.

[0159] A number of possible routes to chemoresistance remain. Recent reports have shown that LOF mutations affecting multiple epigenetic regulators across multiple malignancies can increase the tolerance of cells to environmental stress, a characteristic that may not read out in standard in vitro culture conditions (36). Alternatively, a prominent phenotypic finding in the model described herein was retardation of cell cycle progression, which was demonstrable both transcriptionally and in in-vitro assays, and potentially resulted in the relatively delayed engraftment of 697KIcells in NSG mice. It is therefore possible that slower cell-cycle kinetics could result in relative resistance to predominantly S-phase targeting cytotoxic chemotherapy in patients, as has been shown in pre-clinical models of chemoresistance in B-ALL (37). The mechanism underlying delayed cell cycle progression is likely to be multifactorial, but it is noted recent work implicating CREBBP / EP300 as key mediators maintaining enhancer acetylation marks during cell division (38). Thus, a failure to efficiently reconstitute the chromatin enhancer landscape during mitosis could plausibly result in delayed cell-cycle progression and may also explain why genetic or pharmacologically-induced loss of CREBBP function exerts a more potent phenotype (5) than complete loss of CREBBP protein, through steric competition with residual compensatory EP300 activity.

[0160] It is shown herein that BCL2 inhibition results in programmed cell death through ferroptosis in the context of CREBBP loss of function. Unlike apoptosis, ferroptosis is not mediated by a defined biochemical pathway, rather is the output of a combination of underlying metabolic state, ROS scavenging pathways and lipid composition (39). It has long been known that early B-cell progenitors are exquisitely sensitive to redox balance (40) and recent genetic perturbation data has highlighted a specific propensity for ferroptotic cell death in B-ALL cell lines (41). Reflecting its pleiotropic role in biology (42), how CREBBP loss alters this balance is likely to be multifactorial, including by direct transcriptional changes, as well as posttranslational acetylation of proteins affecting metabolism and redox balance. The functional experiments described herein show significant changes in glycolytic and mitochondrial metabolism both during the acquisition of Venetoclax sensitivity and resistance. In contrast with studies in AML, it is shown herein that there is an association between enhanced metabolic state and BCL2 dependence, likely relating to a cell-state specific susceptibility to oxidative stress (24, 41).

[0161] It has also been demonstrated that small molecule inhibition of CREBBP function, using either the pre-clinical HAT inhibitor A485 or the early-phase CREBBP / EP300-specific bromodomain inhibitor Inobrodib, can sensitize a range of B-ALL cell lines to Venetoclax, potentially widening the paradigm of the combination described herein to multiple ALL genotypes. Importantly, the clinically-actionable combination of Venetoclax and Inobrodib was tolerable and highly efficacious in in-vivo models. Methods Cell lines

[0162] B-ALL cell lines 697, REH and NALM6 cell lines were purchased from DSMZ. RS411, KOPN8 and SUPB15 were provided by Prof. Owen Williams (UCL, UK).697, REH, KOPN8, RS4-11 were cultured at 37˚C with 5% CO2 in IMDM (ThermoFisher, 12440053) supplemented with 10% v / v heat-inactivated fetal bovine serum (HI-FBS) (Sigma Aldrich, F7524), Penicillin-Streptomycin 100U / ml (Sigma Aldrich, P0781) and 1% v / v L-glutamine (Sigma Aldrich, G7513). NALM6 and SUPB15 were cultured in IMDM 20% serum. HEK-293T cells were maintained in DMEM (Sigma D0819), 10% FBS, L-glutamine, and penicillin / streptomycin. Cell lines were tested for mycoplasma by PCR (CSCI Core Facility) and STR genotyped using Promega Powerplex 16-HS kit (performed by Genetica LabCorp). Cells were routinely passaged to 1x106 / ml every 2-4 days. Venetoclax-resistant 697WTand 697KIcells were generated over 10 weeks by exposure to increasing concentrations of Venetoclax (Selleckchem, S8048) until stable growth in 2000nM Venetoclax. Resistant cells were washed to remove Venetoclax at least 24 hours prior to functional experiments. Generation of CREBBP Mutant Lines

[0163] 697 and REH cells were lentivirally-transfected with Cas9, single cell cloned and tested for high Cas9 activity by GFP / BFP reporter assay as previously described (44). CREBBPR1446Cgene-editing reagents were introduced by nucleofection (Nucleofector II, Kit R, Lonza) consisting of a gRNA / Cas9 RNP complex (IDT 1072532, 1081060: target sequenceCATGGTAAACGGCTGTGCGG), two AltR ssDNA Ultramers silently mutating the PAM sequence with or without insertion of the R1446C mutation (50:50 mix: T*T*CACATACTCTAAATATCCAATAAGGATCTCATGGTAAACGGCTGTGCAGAGACAAC GTGGCCGGAAGAAATGAATACTATCCAGATAAGAAATGTA*C*A; T*T*CACATACTCTAAATATCCAATAAGGATCTCATGGTAAACGGCTGTGCGGAGACAAC GTGGCCGGAAGAAATGAATACTATCCAGATAAGAAATGTA*C*A), 3mM homology directed repair enhancer (IDT, 1081072) and IDT nucleofection enhancer oligonucleotide (IDT 1075915) according to manufacturer’s protocol. At 48h ATTO550+vesingle cells were FACS sorted into 96-well plates (BD FACSAria Fusion cell sorter) and, upon growth, colonies screened for successful editing by HpyCH4V amplicon digestion (F:GAGCACCTGGAAAGAGGAGC; R:CCCACAGGCGTGTGTACATT). Clones were validated by Sanger sequencing of the bulk screening amplicon and 10xTOPO-TA-cloned PCR products (ThermoFisher, K4575J10). MTS

[0164] All drugs were purchased from Selleckchem, except A1155463 (Cayman Chemicals), and reconstituted to 10mM in DMSO.72-hour viability assays were performed using CellTiter 96® AQueousOne Solution Cell Proliferation Assay (Promega, G3580) following manufacturer recommendations and 490nM absorbance measured using a CLARIOstar plate reader (BMG Labtech). Drug synergy scores were calculated using SynergyFinder web tool (https: / / synergyfinder.fimm.fi)(45). A peak ZIP threshold of +10 for synergism was defined. Mitochondrial Depolarization using JC1

[0165] Mitochondrial depolarisation was assessed on cells exposed to Venetoclax for 24h using the MitoProbeTMJC-1 assay kit (ThermoFisher, M34152) following manufacturer recommendations. Cells were analysed by flow cytometry (BD Fortessa) for 488nm-excitied 530 / 30 filter signal. Viable cells were gated by DAPI (BD, 564907) exclusion. During flow cytometry cells were routinely handled in DPBS (Gibco, 14190-094) 2% HI-FBS and 2mM EDTA (Invitrogen, 15575038). Annexin V Staining by Flow Cytometry

[0166] Annexin V externalization was assessed using anti-Annexin V-APC kit from (eBioscienceTM88-8007-74) following manufacturer protocol. Briefly, after incubation with drug / vehicle, cells were washed in DPBS, resuspended in Annexin binding buffer andincubated with 1 / 40 Annexin V-APC dilution for 15min at room temperature (RT) in the dark. Cell pellet was resuspended in 7-AAD (BD, 559925) diluted 1 / 100 in 1x binding buffer and analysed by flow cytometry. Western Blot

[0167] Protein lysates were obtained from 1x106cells after resuspending the pelleted cells in 50µl boiling lysis buffer (31mg / ml DTT, 10% SDS, 10% Glycerol, 1% Bromophenol blue and 12.5% 1MTris-HCL). Cell lysates were separated by SDS-PAGE electrophoresis (NuPAGE 3- 8%TRIS acetate protein gels (Invitrogen) or 4-20% Mini-PROTEAN TGX protein gel (Bio- Rad)). Precision-plus protein dual color ladder (Bio-Rad, #1610374) or HiMark™ Pre-stained Protein Standard (Invitrogen, LC5699) were used as a size marker. Proteins were transferred to a methanol pre-activated membrane (Merck, IPFL00010) and then blocked with 5% BSA (ThermoFisher, BP1600) or powdered milk in TBS-0.05% Tween20 (TBST) before proceeding to antibody staining. Primary and secondary antibodies were diluted in 5% BSA or milk in TBST. Primary antibodies were incubated overnight at 4oC or 1h at RT; secondary antibodies were incubated for 1h at RT. Washes were performed with TBST. Membrane fluorescence was acquired using Odyssey imager (LI-COR).

[0168] Antibodies used: Caspase 9 / PARP (Abcam, Ab136812), BCL2 (Abcam, ab32124), β-Tubulin (Sigma-Aldrich, T8328), CREBBP (Santa Cruz biotechnology, SC369) and Vinculin (Santa Cruz biotechnology, sc25336). Lentiviral Production

[0169] Lentiviral vectors were produced in HEK-293T cells by co-transfection of the previously sequenced vector constructs with psPAX and pMDG.2 packaging plasmids using Trans-IT LT-1 (Mirus, MIR2700). HEK293T cells were incubated overnight at 37°C and the next day medium was changed for IMDM 10% HI-FBS. Viral particles were harvested at 48 and 72h by centrifugation and 45μm filtration.250µl of viral particles were used to transfect 1x106B-ALL cells by spinoculation (900g for 2h at 32°C) in the presence of 10μg / ml Polybrene (sc-134220). Subsequently, the cultures were diluted in fresh media and washed three times the following morning.

[0170] Transfection efficiencies were assessed by flow cytometric reporters and transfected cells selected using either 0.5μg / ml of Puromycin (Gibco) for three days or FACS cell sorting as appropriate to achieve >90% transfection.shRNA Knockdown of BCL2

[0171] Doxycycline-inducible reported shRNA knockdown constructs (pLT3GEPIR_GFP / Cherry_Stuffer) were provided by Dr Thomas Mercher (originally from Prof Johannes Zuber), EcoRI / XhoI double digested and the vector backbone purified by gel purification as previously described (25). BCL2 targeting shRNAs were synthesized as Ultramers (IDT) based on the following recommended sequences (BCL2_1: TGCTGTTGACAGTGAGCGCCCGGGAGATAGTGATGAAGTATAGTGAAGCCACAGATGT ATACTTCATCACTATCTCCCGGTTGCCTACTGCCTCGGA; BCL2: TGCTGTTGACAGTGAGCGCGAGGATCATGCTGTACTTAAATAGTGAAGCCACAGATGTA TTTAAGTACAGCATGATCCTCTTGCCTACTGCCTCGGA; Renilla: TGCTGTTGACAGTGAGCGCAGGAATTATAATGCTTATCTATAGTGAAGCCACAGATGTA TAGATAAGCATTATAATTCCTATGCCTACTGCCTCGGA) (25). Complementary oligonucleotides were resuspended to 500ng / µl, boiled and hybridized, ligated using T4 ligase (NEB) (5ng insert:50ng vector) and transformed into chemically-competent E. coli. Bacterial colonies were screened by PCR (F:CTCGACTAGGGATAACAGGG R:CAAAGAGATAGCAAGGTATTCAG) and successful plasmid clones miniprepped (Qiagen) and sequence verified by Sanger sequencing. Cells were selected with puromycin 0.5μg / ml. Cells were incubated with 500ng / ml of doxycycline and mCherry positive cells FACS sorted to confirm knock-down by qPCR and western blot.

[0172] Competitive co-culture assays were performed by mixing an equal number of Renilla shRNA-GFP and one of two BCL2 shRNA-mCherry transfected 697WTor 697KIcells in triplicate. BCL2 knockdown was induced with doxycycline and aliquots of cell culture analysed daily by flow cytometry. Results are presented as the ratio of 697KICherry:GFP events normalized to the mean ratio of 697WTCherry:GFP events. Gene expression by Reverse Transcriptase Quantitative PCR (RT-qPCR)

[0173] Total RNA was extracted using the RNeasy kit (Qiagen) and 1μg was used to synthesize cDNA using TaqMan Reverse transcription (Applied Biosystems, N8080234) following manufacturer’s instructions. RT-qPCR was carried out with SYBR Green PCR master mix (Agilent, 600882) on cDNA (diluted 1:5 in water). The expression level of RNA was calculated using the standard curve method, normalized to the expression of GAPDH. Primer pairs: BCL2F:ATTGGGAAGTTTCAAATCAGC; BCL2R:TGCATTCTTGGACGAGGG; GAPDHF:CCACATCGCTCAGACACCAT; GAPDHR:CCAGGCGCCCAATACG.Seahorse Metabolic Assays

[0174] Oxphos and glycolysis assays were performed using the Seahorse XFe96 analyser (Agilent) according to manufacturer’s instructions. Briefly, cells were analysed at 48-72h post passage and in drug concentrations / DMSO vehicle as described herein. For glycostress experiments, cells were harvested and counted in Seahorse XF base medium, pH7.4 supplemented with 2mM L-Glutamine (Sigma Aldrich, G7513). The final concentration of the injected drugs in the glycostress test is 10mM glucose (Sigma, G7021), 1µM Oligomycin A and 50mM of 2-deoxyglucose (2-DG) (Sigma, D3179). For mitostress tests, Seahorse XF base media was supplemented with 2mM L-Glutamine, 10mM Glucose and 1mM Sodium pyruvate (Sigma, S8636). The final concentration of the injected drugs was 1µM Oligomycin, 1µM FCCP supplemented with 1µM Sodium pyruvate and 1µM Antimycin. For experiments using Venetoclax or A485 pretreatment drug exposure continued in the Seahorse media. Cells were plated at a density of 70000 cells / well in XFp tissue culture plates previously coated with CellTak (Corning, 354240) and incubated for 45mins at 37oC in a CO2-free incubator prior to analysis. Lipid Peroxidation Assays

[0175] Lipid peroxidation was assessed using the lipid peroxidation sensor BODIPYC11- 581 / 591 (ThermoFisher, D3861) according to manufacturer’s instructions. Briefly, cells were washed in DPBS and stained with 4µM BODIPY dye for 30 min at 37oC with gentle shaking in the dark. After washing with DPBS, cells were analysed by flow cytometry. Replicate data is presented as the ratio of 488nm-excited 530 / 30 (“FL1”) to 610 / 20 (“FL3”) filtered signal. RNA Sequencing Library Preparation

[0176] 697WTand 697KItreated with 20nM Venetoclax or DMSO vehicle for 24h (Fig 4) were harvested, washed and RNA was extracted from 4x106cells using the RNeasy kit (Qiagen) (performed in triplicate). Libraries were prepared using NEBNext Poly(A) mRNA magnetic Isolation module (NEB, E7490) starting with 1µg DNA-free RNA, as per manufacturer’s instructions. RNA fragmentation, double-strand cDNA synthesis, end repair, adapter ligation and PCR amplification (9 cycles) was performed using NEBNext Ultra II directional RNA library prep kit for Illumina protocol (NEB, E7760) according to manufacturer’s protocol. Library quality and molarity was measured by Qubit (ThermoFisher) and TapeStation (Agilent).Samples were 50-bp paired-end sequenced on the NovaSeq (Illumina) instrument (CRUK CI Genomics Core Facility). RNAseq analysis

[0177] The quality of the paired-end RNA-seq reads were assessed using FastQC (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). All RNA-seq libraries were found to be above the minimum quality thresholds across quality control metrics. Adapter sequences were trimmed using TrimGalore package (https: / / github.com / FelixKrueger / TrimGalore), and were next mapped against the human genome version 38 (Hg38) using STAR version=2.7.10a (48). Only uniquely-mapping high confidence reads (flags NH:i:1 and MAPQ=255) were retrieved and used for transcriptome quantification with featureCounts version 2.0.1 (49). Genes with a minimum of 10 reads or more in a minimum of two samples were considered for all downstream analysis. Differential expression analysis was based on either a single factor model (~ genotype / treatment / sensitivity) or an interaction model when two factors were considered (~ genotype * treatment), and the interaction term was used to determine the difference of the effect of treatment across genotypes. The interaction model was chosen over the additive model for two-factor analysis of differential expression based on the clustering of normalised RNA-seq libraries on a PCA plot and the number of statistically significant differentially- expressed genes across both models using likelihood-ratio test (LRT). Differential expression analysis was conducted using gene level transcriptomic reads using the Bioconductor package DESeq2 (50). KEGG Pathway enrichment, Gene Set Enrichment Analyses (GSEA) and Gene Ontology (GO) Analyses were all performed using the R package ‘clusterProfiler’ version 4.4.4 (51). A minimum p-value (corrected for multiple testing) and FDR of 0.05 were used across all tests to establish statistical significance. In Vivo dosing studies

[0178] For the in vivo dosing experiments in Figure 6A-F, 0.25x106luciferase expressing cells (isogenic cell lines 697WT& 697KI) were injected by tail vein injection into sub-lethally (2Gy) irradiated 13-15 week-old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) mice. Mice received a daily dose of Venetoclax (100mg / kg)(LC Labs) or vehicle control (15% Kolliphor HS15 (Sigma 42966), 60% Phosal propylene glycol (MP, 151957) and 10% ethanol), by means of Oral Gavage (FTP-20-30, INSTECH) for thirty days or until the experimental endpoints were reached. Disease progression was tracked by IVIS bioluminescence imaging (PerkinElmer); in brief, D-luciferin (Perkin Elmer, 122799) was administered by intraperitoneal(IP) injection (10µl / g body weight of a 15mg / ml solution) followed by inhalation anesthesia (isoflurane) and IVIS bioluminescence imaging (~10 minutes post-luciferin injection). Tumor burden was quantified using Living Image Software (v4.7.2, PerkinElmer).

[0179] For the in vivo dosing experiments in Figure 6G, 0.25x106697WTcells were injected by tail vein injection into sub-lethally (2Gy) irradiated 11-13 week-old old male NSG mice. Mice received a daily dose of Venetoclax (100mg / kg) and / or Inobrodib (20mg / kg) or vehicle control (as above), by means of oral gavage until the experimental endpoints were reached.

[0180] Mice were housed in a pathogen-free animal facility and were allowed unrestricted access to food and water. All experiments were conducted under a UK Home Office project (under the Animals (Scientific Procedures) Act 1986, Amendment Regulations (2012)) and following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body. Statistics

[0181] Flow cytometric data was analysed using FlowJo. Statistical analysis was performed in GraphPad Prism-v9 and Microsoft Excel. Seahorse data was processed on Wave software. Significance tests are stated in figure legends. Significance of survival outcomes was calculated using Mantel Cox log-rank test. PART B 697KI cells are sensitised to chemically diverse BCL2 inhibitors

[0182] Sonrotoclax (BCL2 only) (MedChemExpress) was reconstituted to 10mM in DMSO.72-hour viability assays were performed using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, G3580) following manufacturer recommendations and 490nM absorbance measured using a CLARIOstar plate reader (BMG Labtech).

[0183] The results are shown in Figure 7. Sonrotoclax confirms the sensitivity to BCL2 inhibition, with significant reductions in IC50 in 697KIcells compared to 697WT. Ferroptosis is toxic in Ven resistant B-ALL cells

[0184] Venetoclax-resistant 697WT-Resand 697KI-Rescells were generated in parallel, by escalated exposure to Venetoclax, to generate lines capable of proliferating in 2000nM Venetoclax (the IC50 dose of 697WT). We found a marked induction of Erastin sensitivity upon acquisition of Venetoclax resistance in both resistant cell lines (see Figure 8A). Moreover,co-exposure of 697WT, 697WT-Res and 697KI-Res cells to increasing doses of Venetoclax and / or Erastin demonstrated pharmacological synergy, indicating a potential route to both overcome and prevent Venetoclax resistance (see figure 8B). References 1. Sellar RS, Rowntree C, Vora AJ, Furness CL, Goulden N, Mitchell C, et al. Relapse in teenage and young adult patients treated on a paediatric minimal residual disease stratified ALL treatment protocol is associated with a poor outcome: results from UKALL2003. Br J Haematol.2018;181(4):515-22. 2. Marks DI, Clifton-Hadley L, Copland M, Hussain J, Menne TF, McMillan A, et al. In- vivo T-cell depleted reduced-intensity conditioned allogeneic haematopoietic stem-cell transplantation for patients with acute lymphoblastic leukaemia in first remission: results from the prospective, single-arm evaluation of the UKALL14 trial. Lancet Haematol. 2022;9(4):e276-e88. 3. Pasqualucci L, Dominguez-Sola D, Chiarenza A, Fabbri G, Grunn A, Trifonov V, et al. Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature. 2011;471(7337):189-95. 4. Horton SJ, Giotopoulos G, Yun H, Vohra S, Sheppard O, Bashford-Rogers R, et al. Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors. Nat Cell Biol.2017;19(9):1093-104. 5. Mullighan CG, Zhang J, Kasper LH, Lerach S, Payne-Turner D, Phillips LA, et al. CREBBP mutations in relapsed acute lymphoblastic leukaemia. Nature.2011;471(7337):235- 9. 6. Brady SW, Roberts KG, Gu Z, Shi L, Pounds S, Pei D, et al. The genomic landscape of pediatric acute lymphoblastic leukemia. Nat Genet.2022;54(9):1376-89. 7. Holmfeldt L, Wei L, Diaz-Flores E, Walsh M, Zhang J, Ding L, et al. The genomic landscape of hypodiploid acute lymphoblastic leukemia. Nat Genet.2013;45(3):242-52. 8. Inthal A, Zeitlhofer P, Zeginigg M, Morak M, Grausenburger R, Fronkova E, et al. CREBBP HAT domain mutations prevail in relapse cases of high hyperdiploid childhood acute lymphoblastic leukemia. Leukemia.2012;26(8):1797-803. 9. Li B, Brady SW, Ma X, Shen S, Zhang Y, Li Y, et al. Therapy-induced mutations drive the genomic landscape of relapsed acute lymphoblastic leukemia. Blood.2020;135(1):41-55. 10. Waanders E, Gu Z, Dobson SM, Antic Z, Crawford JC, Ma X, et al. Mutational landscape and patterns of clonal evolution in relapsed pediatric acute lymphoblastic leukemia. Blood Cancer Discov.2020;1(1):96-111.11. Gao C, Liu SG, Lu WT, Yue ZX, Zhao XX, Xing TY, et al. Downregulating CREBBP inhibits proliferation and cell cycle progression and induces daunorubicin resistance in leukemia cells. Mol Med Rep.2020;22(4):2905-15. 12. Chougule RA, Shah K, Moharram SA, Vallon-Christersson J, Kazi JU. Glucocorticoid- resistant B cell acute lymphoblastic leukemia displays receptor tyrosine kinase activation. NPJ Genom Med.2019;4:7. 13. Dixon ZA, Nicholson L, Zeppetzauer M, Matheson E, Sinclair P, Harrison CJ, et al. CREBBP knockdown enhances RAS / RAF / MEK / ERK signaling in Ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response. Haematologica. 2017;102(4):736-45. 14. Lamble AJ, Hagiwara K, Gerbing RB, Smith JL, Kolekar P, Ries RE, et al. CREBBP alterations are associated with a poor prognosis in de novo AML. Blood.2023;141(17):2156- 9. 15. Liu Y, Zheng R, Liu Y, Yang L, Li T, Li Y, et al. An easy-to-use nomogram predicting overall survival of adult acute lymphoblastic leukemia. Front Oncol.2022;12:977119. 16. Qu X, Li H, Braziel RM, Passerini V, Rimsza LM, Hsi ED, et al. Genomic alterations important for the prognosis in patients with follicular lymphoma treated in SWOG study S0016. Blood.2019;133(1):81-93. 17. Malinowska-Ozdowy K, Frech C, Schonegger A, Eckert C, Cazzaniga G, Stanulla M, et al. KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia. Leukemia.2015;29(8):1656-67. 18. Meyer SN, Scuoppo C, Vlasevska S, Bal E, Holmes AB, Holloman M, et al. Unique and Shared Epigenetic Programs of the CREBBP and EP300 Acetyltransferases in Germinal Center B Cells Reveal Targetable Dependencies in Lymphoma. Immunity.2019;51(3):535-47 e9. 19. Lord CJ, Quinn N, Ryan CJ. Integrative analysis of large-scale loss-of-function screens identifies robust cancer-associated genetic interactions. Elife.2020;9. 20. Dutto I, Scalera C, Prosperi E. CREBBP and p300 lysine acetyl transferases in the DNA damage response. Cell Mol Life Sci.2018;75(8):1325-38. 21. Diaz-Flores E, Comeaux EQ, Kim KL, Melnik E, Beckman K, Davis KL, et al. Bcl-2 Is a Therapeutic Target for Hypodiploid B-Lineage Acute Lymphoblastic Leukemia. Cancer Res. 2019;79(9):2339-51. 22. Roca-Portoles A, Rodriguez-Blanco G, Sumpton D, Cloix C, Mullin M, Mackay GM, et al. Venetoclax causes metabolic reprogramming independent of BCL-2 inhibition. Cell Death Dis.2020;11(8):616.23. Pollyea DA, Stevens BM, Jones CL, Winters A, Pei S, Minhajuddin M, et al. Venetoclax with azacitidine disrupts energy metabolism and targets leukemia stem cells in patients with acute myeloid leukemia. Nat Med.2018;24(12):1859-66. 24. Lagadinou ED, Sach A, Callahan K, Rossi RM, Neering SJ, Minhajuddin M, et al. BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells. Cell Stem Cell.2013;12(3):329-41. 25. Fellmann C, Hoffmann T, Sridhar V, Hopfgartner B, Muhar M, Roth M, et al. An optimized microRNA backbone for effective single-copy RNAi. Cell Rep.2013;5(6):1704-13. 26. Zhang J, Vlasevska S, Wells VA, Nataraj S, Holmes AB, Duval R, et al. The CREBBP Acetyltransferase Is a Haploinsufficient Tumor Suppressor in B-cell Lymphoma. Cancer Discov.2017;7(3):322-37. 27. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell.2012;149(5):1060-72. 28. Zhao J, Jia Y, Mahmut D, Deik AA, Jeanfavre S, Clish CB, et al. Human hematopoietic stem cell vulnerability to ferroptosis. Cell.2023;186(4):732-47 e16. 30. Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman DJ, et al. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature.2007;447(7146):864-8. 31. Zou Y, Henry WS, Ricq EL, Graham ET, Phadnis VV, Maretich P, et al. Plasticity of ether lipids promotes ferroptosis susceptibility and evasion. Nature.2020;585(7826):603-8. 32. Herling CD, Abedpour N, Weiss J, Schmitt A, Jachimowicz RD, Merkel O, et al. Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia. Nat Commun.2018;9(1):727. 33. Shoshan-Barmatz V, Shteinfer-Kuzmine A, Verma A. VDAC1 at the Intersection of Cell Metabolism, Apoptosis, and Diseases. Biomolecules.2020;10(11). 35. Place AE, Karol SE, Forlenza CJ, Gambart M, Cooper TM, Fraser C, et al. Pediatric Patients with Relapsed / Refractory Acute Lymphoblastic Leukemia Harboring Heterogeneous Genomic Profiles Respond to Venetoclax in Combination with Chemotherapy. Blood. 2020;136. 36. Loukas I, Simeoni F, Milan M, Inglese P, Patel H, Goldstone R, et al. Selective advantage of epigenetically disrupted cancer cells via phenotypic inertia. Cancer Cell. 2023;41(1):70-87 e14. 37. Turati VA, Guerra-Assuncao JA, Potter NE, Gupta R, Ecker S, Daneviciute A, et al. Chemotherapy induces canalization of cell state in childhood B-cell precursor acute lymphoblastic leukemia. Nat Cancer.2021;2(8):835-52.38. Kikuchi M, Morita S, Wakamori M, Sato S, Uchikubo-Kamo T, Suzuki T, et al. Epigenetic mechanisms to propagate histone acetylation by p300 / CBP. Nat Commun. 2023;14(1):4103. 39. Dixon SJ, Pratt DA. Ferroptosis: A flexible constellation of related biochemical mechanisms. Mol Cell.2023;83(7):1030-42. 40. Borzillo GV, Ashmun RA, Sherr CJ. Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes. Mol Cell Biol.1990;10(6):2703-14. 41. Lalonde ME, Sasseville M, Gelinas AM, Milanese JS, Beland K, Drouin S, et al. Genome-wide CRISPR screens identify ferroptosis as a novel therapeutic vulnerability in acute lymphoblastic leukemia. Haematologica.2023;108(2):382-93. 42. Blobel GA. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood.2000;95(3):745-55. 43. Brooks N, Knurowski T, Hughes A, Clegg K, West W, Pegg NA, et al. CCS1477, a Novel p300 / CBP Bromodomain Inhibitor, Enhances Efficacy of Azacitidine and Venetoclax in Pre-Clinical Models of Acute Myeloid Leukaemia and Lymphoma. Blood.2021;138. 44. Tzelepis K, Koike-Yusa H, De Braekeleer E, Li Y, Metzakopian E, Dovey OM, et al. A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia. Cell Rep.2016;17(4):1193-205. 45. Zheng S, Wang W, Aldahdooh J, Malyutina A, Shadbahr T, Tanoli Z, et al. SynergyFinder Plus: Toward Better Interpretation and Annotation of Drug Combination Screening Datasets. Genomics Proteomics Bioinformatics.2022;20(3):587-96. 46. Sakaue-Sawano A, Kobayashi T, Ohtawa K, Miyawaki A. Drug-induced cell cycle modulation leading to cell-cycle arrest, nuclear mis-segregation, or endoreplication. BMC Cell Biol.2011;12:2. 47. Sakaue-Sawano A, Ohtawa K, Hama H, Kawano M, Ogawa M, Miyawaki A. Tracing the silhouette of individual cells in S / G2 / M phases with fluorescence. Chem Biol. 2008;15(12):1243-8. 48. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics.2013;29(1):15-21. 49. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics.2014;30(7):923-30. 50. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol.2014;15(12):550. 51. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS.2012;16(5):284-7.52. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC- 1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet.2003;34(3):267-73. 53. Dixon SJ, Patel DN, Welsch M, Skouta R, Lee ED, Hayano M, et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife.2014;3:e02523. 54. Montibeller L, de Belleroche J. Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) are characterised by differential activation of ER stress pathways: focus on UPR target genes. Cell Stress Chaperones.2018;23(5):897-912.

Claims

CLAIMS 1. A combination comprising: a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof; and a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof; for use in the treatment of acute lymphoblastic leukaemia (ALL).

2. A combination according to claim 1, wherein the BCL2 inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), obatoclax (GX15-070), oblimersen sodium (G3139), HA14-1, sabutoclax, S55746 (S055746, BCL201), gambogic acid, lisaftoclax (APG2575), sonrotoclax (BGB-11417), AZD4320, AZD-0466 and AT-101, or pharmaceutically acceptable salts thereof.

3. A combination according to claim 1 or claim 2, wherein the BCL2 inhibitor is selected from the group consisting of venetoclax (ABT-199), sonrotoclax (BGB-11417), or navitoclax (ABT-263), or pharmaceutically acceptable salts thereof.

4. A combination according to any one of the preceding claims, wherein the BCL2 inhibitor is venetoclax or a pharmaceutically acceptable salt thereof.

5. A combination according to any one of the preceding claims, wherein the CREBBP and / or EP300 inhibitor is selected from the group consisting of inobrodib, A-485, EP31670, CBP30, GNE-781, JQAD1, or pharmaceutically acceptable salts thereof.

6. A combination according to any one of the preceding claims, wherein the CREBBP and / or EP300 inhibitor is inobrodib or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical product for use in the treatment of ALL comprising a combination according to any one of claims 1 to 6.

8. A pharmaceutical composition product for use in the treatment of ALL comprising a combination according to any one of claims 1 to 6, and one or more pharmaceutically acceptable excipients.

9. A combination according to any one of claims 1 to 6, a pharmaceutical product accoridng to claim 7, or a pharmaceutical composition according to claim 8, for use in: (i) the treatment of B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia;(ii) the treatment of B-ALL, BCP-ALL, or T-ALL; (iii) the treatment of B-ALL; (iv) the treatment of BCP-ALL; (v) the treatment of T-ALL; or (vi) the treatment of lymphoblastic leukaemia.

10. A BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a BCL2 inhibitor or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients, for use in the treatment of ALL, wherein the BCL-2 inhibitor, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered in combination with a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof.

11. A BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use in the treatment of ALL according to claim 10, wherein: the BCL2 inhibitor is selected from one of the following options: (i) venetoclax (ABT-199), navitoclax (ABT-263), obatoclax (GX15-070), oblimersen sodium (G3139), HA14-1, sabutoclax, S55746 (S055746, BCL201), gambogic acid, lisaftoclax (APG2575), sonrotoclax (BGB- 11417), AZD4320, AZD-0466 or AT-101, or pharmaceutically acceptable salts thereof; (ii) venetoclax (ABT-199), navitoclax (ABT-263), obatoclax (GX15-070), or oblimersen sodium (G3139), sonrotoclax (BGB-11417), or pharmaceutically acceptable salts thereof; or (iii) venetoclax, or a pharmaceutically acceptable salt thereof; and the CREBBP and / or EP300 inhibitor is selected from one of the following options:(i) inobrodib, A-485, EP31670, CBP30, GNE-781, or JQAD1, or pharmaceutically acceptable salts thereof; or (ii) inobrodib, or a pharmaceutically acceptable salt thereof.

12. A BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use in the treatment of ALL according to claims 10 or 11, wherein the BCL2 inhibitor is venetoclax, or a pharmaceutically acceptable salt thereof, and the CREBBP and / or EP300 inhibitor is inobrodib, or a pharmaceutically acceptable salt thereof.

13. A BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claims 10, 11 or 12, wherein the BCL2 inhibitor is for use in: (i) the treatment of B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia; (ii) the treatment of B-ALL, BCP-ALL, or T-ALL; (iii) the treatment of B-ALL; (iv) the treatment of BCP-ALL; (v) the treatment of T-ALL; or (vi) the treatment of lymphoblastic leukaemia.

14. A CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a CREBBP and / or EP300 inhibitor or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients, for use in the treatment of ALL, wherein the CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered in combination with a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof.

15. A CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use in the treatment of ALL according to claim 14, wherein: the BCL2 inhibitor is selected from one of the following options:(i) venetoclax (ABT-199), navitoclax (ABT-263), obatoclax (GX15-070), oblimersen sodium (G3139), HA14-1, sabutoclax, S55746 (S055746, BCL201), gambogic acid, lisaftoclax (APG2575), sonrotoclax (BGB- 11417), AZD4320, AZD-0466 and AT-101, or pharmaceutically acceptable salts thereof; (ii) venetoclax (ABT-199), sonrotoclax (BGB-11417), or navitoclax (ABT- 263), or pharmaceutically acceptable salts thereof; or (iii) venetoclax, or a pharmaceutically acceptable salt thereof; and the CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, is selected from one of the following options: (i) inobrodib, A-485, EP31670, CBP30, GNE-781, JQAD1, or pharmaceutically acceptable salts thereof; or (ii) inobrodib, or a pharmaceutically acceptable salt thereof.

16. A CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use in the treatment of ALL according to claim 14 or claim 15, wherein the BCL2 inhibitor is venetoclax, or a pharmaceutically acceptable salt thereof and and the CREBBP and / or EP300 inhibitor is inobrodib, or a pharmaceutically acceptable salt thereof.

17. A CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 14 to 16, wherein the CREBBP and / or EP300 inhibitor is for use in: (i) the treatment of B-ALL, BCP-ALL, T-ALL or lymphoblastic leukaemia; (ii) the treatment of B-ALL, BCP-ALL, or T-ALL; (iii) the treatment of B-ALL; (iv) the treatment of BCP-ALL; (v) the treatment of T-ALL; or (vi) the treatment of lymphoblastic leukaemia.

18. A combination for use according to any one of claims 1 to 6 and 9, a pharmaceutical product for use according to claims 7 and 9, a pharmaceutical composition for use according to claims 8 and 9, a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use according to any one of claims 10 to 13, or a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use according to any one of claims 14 to 17, wherein: (i) the BCL2 inhibitor and the CREBBP and / or EP300 inhibitor are administered simultaneously; (ii) the BCL2 inhibitor and the CREBBP and / or EP300 inhibitor are administered sequentially; (iii) the BCL2 inhibitor and the CREBBP and / or EP300 inhibitor are administered separately; (iv) the CREBBP and / or EP300 inhibitor is administered prior to the BCL2 inhibitor; or (v) a pre-treatment with the CREBBP and / or EP300 inhibitor is administered prior commencing treatment with the BCL2 inhibitor.

19. A combination for use according to any one of claims 1 to 6 and 9, a pharmaceutical product for use according to claims 7 and 9, a pharmaceutical composition for use according to claims 8 and 9, a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use according to any one of claims 10 to 13, or a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use according to any one of claims 14 to 17,wherein the BCL2 inhibitor and the CREBBP and / or EP300 inhibitor are administered either orally, intravenously or by intrathecal injection.

20. A combination for use according to any one of claims 1 to 6 and 9, a pharmaceutical product for use according to claims 7 and 9, a pharmaceutical composition for use according to claims 8 and 9, a BCL2 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use according to any one of claims 10 to 13, or a CREBBP and / or EP300 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use according to any one of claims 14 to 17, wherein the CREBBP and / or EP300 inhibitor is selected from the group consisting of: (i) a selective CREBBP inhibitor; or (ii) a dual CREBBP and EP300 inhibitor.

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