Epichaperome Inhibition Targets Kinase and TP53 Mutant Therapy Resistant AML

The BCL-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, improves outcomes for many patients with AML and is now considered standard of care for patients older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months; 2.5 months after relapse. Mutations of FLT3, PTPN11, and RAS, leading to constitutive activation of cell survival signaling, and mutations of TP53 have been identified as key determinants of low response rates, early relapse and death. Mutant TP53 cells are also characterized by activation of signaling kinases.

The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. Epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, epichaperome inhibition has the potential of eliminating resistant subclones before they become dominant and cause relapse. In contrast to other HSP90 purine analog inhibitors, PU-H71(zelavespib) is a competitive inhibitor specific for the conformationally changed ATP binding site of HSP90 when incorporated into epichaperomes. We here investigate the therapeutic potential of targeting epichaperomes with PU-H71 in TP53 and oncogenic kinase mutant AML.

Western blot analysis showed increased HSP90 and several signaling proteins such as STAT3 and c-Myc in TP53-knockout and mutant Molm13 cells generated by CRISPR/cas-9, compared to isogeneic wild-type (WT) controls. Using fluorochrome-labelled PU-H71 and flow cytometry, we demonstrated the presence of epichaperome in AML and AML stem/progenitor cells with TP53 and/or RAS mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively killed AML cells, including those with acquired resistance to BH3 mimetics and those with various FLT3 mutants. PU-H71 targets AML cells and stem/progenitor cells with TP53 mutations, and prolonged survival of TP53-mutant AML xenograft mice with minimal effects on mouse bone marrow LSK cells and hematopoiesis. PU-H71 increased the expression of pro-apoptotic BIM and the efficacy of VEN in AML cells and AML stem/progenitor cells with TP53 and/or RAS mutations (combination index < 1). Importantly, in a 1,000: 1 mixture of TP53-WT and TP53-R248W mutant isogenic Molm13 cells, MDM2 inhibitor nutlin3a selectively killed TP53-WT but enriched TP53-mutant Molm13 cells, as expected, and VEN treatment also favored the outgrowth of TP53-mutant cells. However, PU-H71 effectively killed TP53-WT and mutant cells (Fig. 1): 10 million TP53-WT cells were reduced to 335 cells, and 10,000 TP53-mutant cells to 26. The activity of PU-H71 against both TP53-WT and mutant AML cells was further enhanced by VEN in vivo in a xenograft model of mixed (10:1) TP53-WT and mutant Molm13 cells.

Our data support the concept that the epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring TP53 and/or oncogenic kinase mutations. Inhibition of epichaperomes by PU-H71 targets AML cells/stem/progenitor cells, enhances VEN activity, and prevents the outgrowth of nutlin- or VEN-resistant TP53-mutant AML cells. This concept has entered clinical evaluation in MPN-derived secondary AML.

Carter:PinotBio: Research Funding; Syndax: Research Funding; Revolution Medicines: Research Funding. Nishida:Daiichi-Sankyo Inc.: Research Funding; Kintor Pharmaceutical: Research Funding. Pemmaraju:stemline: Consultancy; abbvie: Consultancy; immunogen: Consultancy; mustangbio: Research Funding; incyte: Consultancy; novartis: Research Funding; pacylex: Consultancy, Research Funding; samus: Research Funding; daiichi sankyo: Research Funding; cellectis: Research Funding; cellularity: Research Funding. Shpall:Navan: Consultancy; Takeda: Patents & Royalties; axio: Consultancy; adaptimmune: Consultancy; Bayer: Honoraria; Affimed: Other: License agreement; Fibroblasts and FibroBiologics: Consultancy; NY blood center: Consultancy. Chiosis:Samus Therapeutics Inc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Andreeff:Kintor Pharmaceutical: Research Funding; Daiichi-Sankyo Inc.: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; AstraZeneca: Research Funding; NCI: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Medicxi: Consultancy; Reata: Current holder of stock options in a privately-held company; Chimerix: Current holder of stock options in a privately-held company; Glycomimetics: Consultancy; Oncolyze: Current holder of stock options in a privately-held company; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees; Senti Bio: Consultancy, Research Funding; Syndax: Consultancy, Research Funding; Brooklyn ITX: Research Funding; Pinot Bio: Research Funding; Oxford Biomedical UK: Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees.