Selective SMARCA2 Degradation Promotes Leukemic Differentiation and Synergizes with CDK9 Inhibition to Potently Induce Death in Pre-Clinical Models of Acute Myeloid Leukemia
Authors: Fultang Norman , Schwab Ashley M , Johnson Ian , Grego Alexander , Filler Emma , Bachner Carly , Karwoski Jake , Moore Andrew , Bartilomo Ashley , Agarwal Anjana , Osinubi Olusola Peace , Ito Koichi , Aminov Sarah , Sahu Srabani , Verma Amit , Scherle Peggy A , Bhagwat Neha , Heiser Diane 
Published: December 2024

SWI/SNF (BAF) nucleosome remodeling complexes play an important role in regulating gene expression by maintaining chromatin accessibility. Mammalian SWI/SNF complexes contain two mutually exclusive, functionally redundant ATPase subunits, SMARCA2 and SMARCA4. Deregulated SWI/SNF activity has been linked to the pathogenesis of several malignancies including myeloid disorders like AML and MDS.

In AML/MDS, deregulated BAF activity has been associated with differentiation block, allowing the accumulation of immature leukemic stem cells in the myeloid compartment. Small molecule inhibitors targeting both BAF ATPase subunits have shown promising clinical activity, inducing differentiation of leukemic stem cells in AML/MDS patients, with dose-limiting toxicities. In genetic ablation studies, dual loss of SMARCA2 and SMARCA4 has been associated with pronounced cytotoxicity in multiple cell types. Selective ablation of SMARCA2, however, is tolerated due to functional compensation by SMARCA4.

In the present study, we show that PRT3789, a highly selective clinical-stage SMARCA2 degrader, similarly induces differentiation in AML progenitor cells with improved therapeutic window over dual SMARCA2/4 targeted agents, and synergizes with CDK9 inhibition to potently suppress leukemic cell growth in pre-clinical models.

Exposure to low doses of PRT3789 (0.3 to 5 nM) for 3-7 days induced surface CD11b expression in AML cell lines KG1, THP-1, HL-60 and OCI-AML3. Immunoblots confirmed selective degradation of SMARCA2 (DC50 range: 1.0 - 2.3 nM) by PRT3789, with no activity on SMARCA4 (DC50 range: &gt;100 nM), in these cell lines. Selective degradation of SMARCA2 coincided with increased expression of IRF8 and MYC, two key transcriptional regulators of myeloid cell maturation and function, at 72h post-treatment.

Increased MYC expression has been shown to sensitize cells to CDK9 inhibitor-mediated cytotoxicity. Based on this observation, we assessed the combined cytotoxic effect of PRT3789 with PRT2527, a highly selective clinical-stage CDK9 inhibitor, in various cell lines. Pre-treatment with PRT3789 (6 days) followed by brief exposure to PRT2527 (24h) synergistically repressed growth and induced cell death in multiple AML cell lines. In OCIAML3, HL-60 and THP-1 cell line-derived xenografts, PRT3789 combined with PRT2527 to potently repress tumor growth, including tumor regressions, outperforming PRT2527 alone. Following the conclusion of dosing, tumor regrowth in animals co-treated with PRT3789 and PRT2527 was significantly delayed compared to monotherapy groups. In monotherapy, PRT3789 treatment showed minimal tumor growth inhibition, a phenotype typical of differentiation agents in AML. End-of-study flow cytometry analysis confirmed increased surface CD11b, CD64 and CD14 expression on the cells of tumors treated with PRT3789. Taken together, these results suggest PRT3789 induces differentiation in AML cells in vitro and in vivo and combines with PRT2527 to potently induce cell death and inhibit tumor growth.

To evaluate the effects of PRT3789 on primary AML patient samples (N=4, with mutations including CEBPA, TET2, GATA2, NRAS, TP53, NPM1, DNMT3A, WT1), we performed clonogenic assays and FACS analysis for differentiation markers in colonies. We observed decreased leukemic colony growth in all samples after treatment with PRT3789 at nanomolar doses. Degradation of SMARCA2 alone led to increased differentiation as evident from increase in CD11b expression. Healthy donor PBMCs as well as healthy CD34 cells treated with PRT3789 showed no significant changes in differentiation markers and notably, cell viability was maintained during treatment. These data suggest a therapeutic window for PRT3789 treatment in AML patients without targeting healthy cells.

Altogether, these findings highlight the promise of PRT3789 as a differentiation agent in myeloid malignancies, and in combination with PRT2527 for the treatment of relapsed/refractory AML/MDS.

PRT2527 is currently being evaluated in Phase I clinical trials for relapsed/refractory hematologic malignancies (NCT05665530). PRT3789 is currently being evaluated in Phase I clinical trials for advanced or metastatic solid tumors with SMARCA4 mutations (NCT05639751).