Efficacy of Combinations of Targeted Agents Against Dependencies in Mecom Rearranged AML Identified By Unbiased Chemical and CRISPR Screens

The MECOM locus (located at 3q26.2) encodes for the multi-zinc finger domain transcription factor EVI1, which regulates normal hematopoietic stem cell maintenance. EVI1 targets include other major regulators such as ERG and MYC. Aberrant EVI1 overexpression is observed in ~10% of de novo AML, of which approximately half are due to the chromosomal rearrangement involving either inv(3)(q21;q26.2) or t(3;3)(q21;q26.2). This chromosomal rearrangement (3q26.2-r) results in relocation of the distal GATA2 hematopoietic enhancer to EVI1 promoter, which now drives EVI1 expression. In 3q26.2-r AML, co-mutations in addition to GATA2 repression with increased EVI1 activity promote AML progression. This is associated with an aggressive phenotype that is relatively refractory to the available treatment options. Therefore, there is need to develop and test novel targeted therapies with improved efficacy in repressing EVI1 and improving survival of AML patients with EVI1 overexpression.

We used two approaches to identify druggable vulnerabilities in 3q26.2-r AML. We used an unbiased high-throughput drug screen focused on mechanistically annotated drugs (NCATS Mechanism Interrogation Plates [MIPE 5.0] and domain-specific epigenetic-targeted CRISPR screen. In both screens, BRD4 was identified as an absolute dependency in the 3q26.2-r AMLs. This was consistent with our previous report showing that BET inhibitors (BETi) are effective against 3q26.2-r AML cell lines, patient-derived (PD) AML cells and their PDX models (Leukemia 2024; 38: 545-556). In addition to BRD4, the CRISPR screen identified KDM1A (LSD1), p300, and HDAC3 as dependencies in a 3q26.2-r AML cell line (UCSD-AML1), which was further evaluated here. Treatment of 3q26.2-r AML cell lines for 96-hours with ORY001 (LSD1i; 3-100 nM), RGFP966 (HDAC3i; 0.5-10 µM) or GNE-781 (CBP/p300i; 10-1000 nM) induced moderate, dose-dependent cell death, as determined by flow cytometry. However, all three agents, especially LSD1i treatment, caused a marked increase in CD11b and CD86 double-positive cells (50-80%), suggesting that these treatments also induced differentiation of 3q26.2-r AML cell lines. Following these treatments, Western analysis of 3q26.2-r AML cell lines showed a decrease in phosphorylated S6 and EVI1 expressions and an increase in CD11b protein levels. Additionally, the high-throughput NCATS MIPE 5.0 screen identified mTOR, PIK3CA, and XIAP as druggable vulnerabilities in 3q26.2-r AML cells. In follow-up experiments, mivebresib (BETi) dactolisib (BEZ235, mTOR/PI3Ki) and LCL161 (IAPi) induced dose-dependent apoptosis in 3q26.2-r cell lines. In PD AML samples, treatment with mivebresib and dactolisib for 48-hours reduced viability to a greater extent in 3q26.2-r AML, compared to non-3q26.2-r AMLs cells (p < 0.01). Tandem-tag mass spectrometry of a 3q26.2-r AML cell line with gene-set enrichment analysis (GSEA) demonstrated that treatment with mivebresib, dactolisib or LCL161 for 16 hours downregulated MYC-targets gene-set. Mivebresib and dactolisib-treated cells also showed downregulation of cell-cycle related gene-set. These findings were further corroborated by qRT-PCR, Western blot and cell-cycle analyses. Combination of mivebresib (50-250 nM) with dactolisib (50-500 nM) or LCL161 (30-1000 nM) synergistically induced in vitro apoptosis in 3q26.2-r AML cells (delta synergy scores > 1.0). Treatment of the luciferized 3q26.2-r PD AML242 model, engrafted in NSG mice, with mivebresib (0.5 mg/kg, daily 5x per week), dactolisib (20 mg/kg, daily 5x per week) or LCL161 (65 mg/kg, daily 5x per week) reduced AML burden and increased survival compared to vehicle-treated mice (p < 0.05). Importantly, in a separate luciferized 3q26.2-r AML194 PDX model, the combinations also reduced the in vivo AML burden and increased survival of the mice, as compared to treatment with the vehicle control or with single agent alone (p < 0.05). Collectively, these findings show that both CRISPR and chemical screens identified novel druggable targets, as well as helped develop treatments with their targeted agents. These treatments resulted in reduction of EVI1 and c-Myc levels, associated with preclinical differentiation and apoptotic activity in the 3q26.2-r AML cell models. Further in vivo evaluation of the efficacy of BETi or LSD1i-based combinations against 3q26.2-r AML is warranted.

Kadia:Abbvie: Consultancy, Research Funding; Incyte: Research Funding; Servier: Consultancy; Sellas: Consultancy, Research Funding; Regeneron: Research Funding; BMS: Consultancy, Research Funding; Ascentage: Research Funding; Pfizer: Research Funding; Genentech: Consultancy, Research Funding; Rigel: Honoraria; ASTEX: Research Funding; AstraZeneca: Research Funding; Novartis: Honoraria; DrenBio: Consultancy, Research Funding; JAZZ: Research Funding; Amgen: Research Funding; Cellenkos: Research Funding. Daver:FATE Therapeutics: Other: Consulting Fees, Research Funding; Astellas: Consultancy, Research Funding; KITE: Research Funding; Novimmune: Research Funding; Servier: Consultancy, Research Funding; Arog: Consultancy; Trovagene: Research Funding; Celgene: Consultancy; Agios: Consultancy; Shattuck Labs: Consultancy; Syndax: Consultancy; Novartis: Consultancy; Hanmi: Research Funding; Jazz: Consultancy; Trillium: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Menarini Group: Consultancy; Glycomimetics: Research Funding; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Research Funding. DiNardo:ImmuneOnc: Research Funding; Schrodinger: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Other: meetingsupport, Research Funding; Astellas: Consultancy, Honoraria; Gilead: Consultancy; BMS: Consultancy, Honoraria, Research Funding; Notable Labs: Honoraria; Loxo: Research Funding; Amgen: Consultancy; GenMab: Consultancy, Honoraria, Other: data safety board; GSK: Consultancy, Honoraria; Foghorn: Research Funding; AstraZeneca: Honoraria; Cleave: Research Funding; Rigel: Research Funding; Astex: Research Funding; Genetech: Honoraria; Riegel: Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Immunogen: Honoraria; Jazz: Consultancy, Honoraria; Stemline: Consultancy. Sasaki:Pfizer: Consultancy; Daiichi-Sankyo: Consultancy; Novartis: Consultancy, Research Funding; Enliven: Research Funding; Otsuka: Other: Lecture fees; Chugai: Other: Lecture fees.