Efficacy of a Novel BCL-XL Degrader, DT2216, in Preclinical Models of Post-Myeloproliferative Neoplasm Secondary AML

Acute myeloid leukemia (AML) that evolves from myeloproliferative neoplasm (MPN), known as post-MPN secondary (s) AML, has a dismal prognosis, with current treatments failing to extend survival significantly beyond 12 months (Dunbar et. al., Blood 2020). B-cell lymphoma-extra-large (BCL-XL), an anti-apoptotic protein in the BCL-2 family, is overexpressed in MPN patients (Petiti et. al. J Cell Mol Med. 2020). This study investigated the role of BCL-XL in post-MPN sAML and evaluated the efficacy of DT2216 (kindly provided by Dialectic Therapeutics, Inc.), a platelet-sparing BCL-XL proteolysis-targeting chimera (PROTAC), in preclinical models.

To assess BCL-XL expression and dependency in post-MPN sAML, we analyzed the RNA-seq data from the Beat 1.0 AML cohort and Cancer Cell Line Encyclopedia, CRISPR screen data from Public Avana 21Q2 dataset, and multi-omics single cell data from TP53-mutant post-MPN sAML patients (Rodriguez-Meira et. al., Nat Genet. 2023). We found that post-MPN sAML patients exhibited higher expression of BCL2L1 (p=0.0026), which encodes BCL-XL, compared to de novo AML patients. JAK2-mutant AML cell lines, representing cell line models for post-MPN sAML, showed higher BCL2L1 expression (p=0.02) and lower CRISPR gene scores (p=0.02) for BCL2L1 (compared to non-JAK2-mutant AML cell lines), indicating stronger dependence on BCL-XL. Single cell multi-omics data from 9,709 cells corresponding to 13 post-MPN sAML samples revealed that hematopoietic stem and progenitor cells (HSPCs) with elevated BCL2L1 expression were enriched among JAK2/CALR_TP53-mutant HSPCs. These HSPCs exhibited features of both leukemia stem cells and erythroid/megakaryocytic differentiation (calculated scores by the corresponding gene sets).

Using JAK2 wild-type (WT) and JAK2^V617F induced pluripotent stem cell (iPSC)-derived CD34⁺ HSPCs from the same MPN patient, we found significantly higher BCL2L1 expression in JAK2^V617F iPSC-HSPCs compared to JAK2 WT cells (p<0.0001) by qPCR. Knocking out BCL2L1 using CRISPR/Cas9 in JAK2-mutant AML cell lines (SET2 and HEL) halted their proliferation, while silencing BCL2L1 had no impact on non-JAK2-mutant AML cell lines (THP1 and OCI-AML3). BH3 profiling revealed that JAK2^V617F iPSC-HSPCs (compared to JAK2 WT iPSC-HSPCs), JAK2-mutant cell lines and post-MPN sAML primary samples (compared to BCL-2-dependent MOLM13 cells) released more cytochrome C after treatment with the BCL-XL-specific peptide HRK-Y, PROTAC DT2216, and dual BCL-XL/BCL-2 inhibitor ABT-263. In addition, compared to parental (pa) cells, ruxolitinib-resistant (ruxo-re) JAK2-mutant (SET2 and HEL) cells showed profound BCL-XL dependence by BH3 profiling.

We then tested activity of BCL-XL degrader DT2216 in JAK2-mutant AML cell lines, iPSC-HSPCs and post-MPN sAML primary samples by Cell Titer Glo or Colony-forming unit (CFU) assays. DT2216 significantly reduced cell viability in JAK2-mutant AML cell lines (average IC₅₀: 1.6±0.8μM in pa lines, 5.4±3.8 μM in ruxo-re lines at 72 hours) and JAK2-mutant iPSC-HSPCs (IC₅₀: 0.04μM), while WT iPSC-HSPCs were unaffected. Notably, DT2216 also effectively reduced cell viability of CD34⁺ primary samples (n=6, average IC₅₀: 2.2±1.6μM at 24 hours). In SET2 cell line-derived xenograft (CDX) models, DT2216 reduced the leukemic burden and extended the survival compared to the vehicle group (p=0.01), associated with degradation of BCL-XL. Combination of DT2216 and azacytidine (AZA), ruxolitinib or venetoclax demonstrated potent efficacy and synergistic anti-leukemia cell viability in vitro exhibiting combination indexes of <1.0 (by Calcusyn software). Western blotting showed efficient degradation of BCL-XL and activation of apoptosis with DT2216 alone and in combinations. In CFU assays using CD34⁺ cells from three primary post-MPN sAML patients, compared to DMSO, AZA (100 nM) treatment resulted in a 30% reduction in colony count (range 25.7-36.4%, p=0.0008). DT2216 (50 nM) led to a 69% reduction (range 58.3-77.5%, p=0.0003), and the combination of DT2216/AZA yielded a 76% reduction (range 70.9-79.8%, p<0.0001).

Taken together, our findings highlight the survival dependence of post-MPN sAML on anti-apoptotic BCL-XL. The promising efficacy of DT2216, evidenced by reduced cell viability, synergistic effects with AZA, and in vivo activity in CDX mouse model, supports its potential as a therapeutic option for post-MPN sAML.

Rodriguez-Meira:Aletheiomics: Consultancy; Intellectual Ventures: Honoraria. Zheng:Dialectic Therapeutics: Other: Co-founders and shareholder of Dialectic Therapeutics, a company that is developing Bcl-xL PROTACs to treat cancers; Co-inventor of the Bcl-xL PROTACs disclosed in this study. Zhou:Dialectic Therapeutics: Other: Co-founder and shareholder of Dialectic Therapeutics, a company that is developing Bcl-xL PROTACs to treat cancers; Co-inventor of the Bcl-xL PROTACs disclosed in this study. Mead:Alethiomics: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company, Research Funding; Incyte: Consultancy, Honoraria; Galecto: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; GSK: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria; Medscape: Honoraria; Ionis: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Roche: Research Funding. Rampal:Sumitomo Dainippon: Consultancy; Kartos: Consultancy; Servier: Consultancy; Zentalis: Consultancy, Research Funding; Karyopharm: Consultancy; Protagonist: Consultancy; Sierra Oncology/GSK: Consultancy; Constellation/MorphoSys: Consultancy, Research Funding; Jubilant: Consultancy; PharmaEssentia: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Galecto: Consultancy; Ryvu: Research Funding; Disc Medicine: Consultancy; AbbVie: Consultancy; Cogent: Consultancy; Incyte Corporation: Consultancy, Research Funding; CTI BioPharma: Consultancy; Celgene/BMS: Consultancy; Blueprint: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Promedior: Consultancy. Abbas:Ascentage: Research Funding; Blueprint Medicines Corporation: Research Funding; GlaxoSmithKline: Research Funding; Genentech: Research Funding; Alamar Biosciences: Honoraria; Illumina: Honoraria, Other: Inkind Support, Research Funding; Molecular Partners: Consultancy; Enzyme By Design: Research Funding. Pemmaraju:Bristol-Myers Squibb: Consultancy; Springer Science + Business Media: Honoraria; Stemline Therapeutics: Honoraria, Other: Travel Expenses, Research Funding; Aptitude Health: Honoraria; Roche Molecular Diagnostics: Honoraria; Mustang Bio: Honoraria, Other: Travel Expenses, Research Funding; LFB Biotechnologies: Honoraria; Incyte: Honoraria; Protagonist Therapeutics: Consultancy; Celgene: Honoraria, Other: Travel Expenses; ClearView Healthcare Partners: Consultancy; Neopharm: Honoraria; Blueprint Medicines: Consultancy, Honoraria; CareDx: Honoraria; Pacylex: Consultancy; DAVA Oncology: Honoraria, Other: Travel Expenses; Novartis: Honoraria, Research Funding; CTI BioPharma: Consultancy; Immunogen: Consultancy; Plexxikon: Research Funding; Triptych Health Partners: Consultancy; Affymetrix/Thermo Fisher Scientific: Research Funding; Cellectis: Research Funding; Daiichi Sankyo: Research Funding; Samus Therapeutics: Research Funding; Blueprint Medicines OncLive PeerView Institute for Medical Education: Consultancy, Other: advisory board; Astellas: Consultancy; AbbVie: Honoraria, Other: Travel Expenses, Research Funding; ASH Committee on Communications ASCO Cancer.NET Editorial Board: Other: Leadership; Karger Publishers: Other: Licenses; National Institute of Health/National Cancer Institute (NIH/NCI): Research Funding; HemOnc Times/Oncology Times: Other: uncompensated. Konopleva:Sellas: Consultancy; AstraZeneca: Consultancy, Other: clinical trials, Research Funding; Sanofi Aventis: Consultancy, Other: clinical trials, Research Funding; Menarini Group: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: clinical trials, Research Funding; Reata Pharmaceutical: Other: IP; Bakx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Redona: Consultancy; ImmunoGen: Research Funding; Janssen: Consultancy, Other: clinical trials; Vincerx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Research Funding; Rafael Pharmaceutical: Research Funding; Immune Oncology: Membership on an entity's Board of Directors or advisory committees; Allogene: Research Funding; MEI Pharma: Consultancy, Research Funding; Pfizer: Other: clinical trials; Auxenion GmbH: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: clinical trials, Research Funding; Dark Blue Therapeutics: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-LaRoche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Other: Clinical Trials; Boehringer: Consultancy; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: clinical trials, Research Funding.