Pharmacodynamic Analysis of Mcl-1 and RNA Pol IISer2 Phosphorylation in Blasts from Patients with Relapsed or Refractory Acute Myeloid Leukemia Treated on a Phase 1 Study of Venetoclax Plus Voruciclib

Background: Patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy are often treated with venetoclax in combination with hypomethylating agents (HMAs) or low dose cytarabine. While these regimens can be effective, resistance frequently develops, and response to subsequent lines of therapy is often poor. Increased survival dependence on an alternative anti-apoptotic protein, myeloid cell leukemia-1 (Mcl-1), has been identified as a key mechanism driving resistance to venetoclax. Voruciclib, an oral inhibitor of cyclin-dependent kinase 9 (CDK9), has been shown to indirectly reduce Mcl-1 transcription and protein expression, enhancing the activity of venetoclax in preclinical models of AML.

Leukemic stem cells (LSCs) have been previously shown to be a key target of 5-AZA/VEN-based therapy, and higher baseline Mcl-1 expression was observed in patients who were non-responders to 5-AZA/VEN. Here, we study patient samples from a phase 1 clinical trial of venetoclax plus voruciclib, which found this combination to be well-tolerated, with potential anti-leukemic activity in relapsed/refractory (R/R) AML. We hypothesized that the pharmacodynamic change in Mcl-1 protein expression in LSCs in response to venetoclax and voruciclib may be associated with clinical response.

Methods: In a phase 1 trial (NCT# 03547115), patients with R/R AML received voruciclib at seven dose levels (50-300 mg) on days 1-14 of a 28-day cycle, with the first cycle starting voruciclib on day 3. Venetoclax was administered daily throughout the cycle, with an initial dose of 100 mg on day 1, followed by 200 mg on days 2-21, and 400 mg on days 22-28. Peripheral blood mononuclear cells (PBMCs) were viably frozen and later thawed in batches for analysis. Flow cytometry was used to measure Mcl-1 protein levels and RNA Pol II^Ser2 phosphorylation in AML blasts at baseline and at specific time points during the first cycle: at 0 and 6 hours on day 1, at 0 and 6 hours on day 3, and at 0 and 6 hours on day 14. The AML blast population was subdivided into LSC-like, non-LSC-like, and mature blasts to assess differential responses. A gating strategy of cell-surface expression of CD64 and CD11b was used to distinguish between mature CD64+CD11b+ blasts and immature blasts. GPR56 expression was included to identify LSCs within the immature blast subpopulation.

Results: Responders (defined as patients who achieved a morphologic leukemia-free state (MLFS), complete remission with partial hematologic recovery (CRh), or complete remission with incomplete hematologic recovery (CRi)) showed a 1.8-fold increase (p=0.0358) in Mcl-1 protein expression following venetoclax treatment and prior to voruciclib initiation on day 3. In the same patients, Mcl-1 returned to baseline after 14 days of combination venetoclax plus voruciclib treatment, a significant decrease from peak Mcl-1 prior to voruciclib initiation (p=0.0090). In contrast, patients with progressive or stable disease had no significant change in Mcl-1 protein expression after initiating venetoclax or the combination of venetoclax plus voruciclib. Responders also had significantly lower RNA Pol II^Ser2 phosphorylation after 14 days of combination treatment compared to baseline (0.7-fold decrease, p=0.0024). Baseline Mcl-1 protein expression was highest in the mature blast population, a 1.3-fold (p=0.0028) difference when compared to LSC-like blasts; however, baseline MCL-1 expression in bulk blasts or subpopulations was not related to clinical response. Patients who achieved CRh or MLFS had the largest fold decrease in Mcl-1 protein expression in LSC-like blasts (0.5 compared to 1.0 in non-responders, p=0.0418), which was detected 6h after voruciclib initiation.

Conclusions: The combination of venetoclax plus voruciclib demonstrated pharmacodynamic activity in AML, and modulation of Mcl-1 protein expression and RNA Pol II phosphorylation, both of which were associated with clinical response. These findings highlight the potential of indirectly targeting Mcl-1 through CDK9i to improve outcomes for patients with AML and underscore the importance of further investigating the mechanisms underlying the efficacy of the combination of venetoclax plus voruciclib in this context.