Efficacy of a Novel BCL-XL Degrader, DT2216, in Preclinical Models of Post-Myeloproliferative Neoplasm Secondary AML
Authors: Wang Zhe , Skwarska Anna , Poigailwar Gowri , Chaudhry Sovira , Rodriguez-Meira Alba , Sui Pinpin , Olivier Emmanuel , Jia Yannan , Ramage Cassandra L. , Zheng Guangrong , Schurer Alexandra , Gritsman Kira , Papapetrou Eirini , Bhalla Kapil N. , Zhou Daohong , Mead Adam J , Rampal Raajit , Tyner Jeffrey W , Abbas Hussein A. , Pemmaraju Naveen , Tatarata Qi Zhang , Konopleva Marina 
Published: December 2024

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 JAK2V617F induced pluripotent stem cell (iPSC)-derived CD34+ HSPCs from the same MPN patient, we found significantly higher BCL2L1 expression in JAK2V617F iPSC-HSPCs compared to JAK2 WT cells (p&lt;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 JAK2V617F 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 &lt;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&lt;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.