Venetoclax Rapidly and Strongly Enhances the Phospholipase C Response to Azacitidine Therapy in Myelodysplastic Syndromes

Background and Rationale. Azacitidine (AZA) is a standard treatment for MDS patients. The addition of Venetoclax (VEN) to AZA is effective in both AML and higher risk MDS (Bazinet A and Montalban Bravo G, Curr Treat Options Oncol, 2022). Previous studies showed that a favorable response to AZA is linked to an increased expression of PLCbeta1 and PLCgamma2, two phospholipases (PLCs) implicated in the regulation of cell proliferation and differentiation (Cocco L et al, J Leuk Biol, 2015). AZA positive effect also resulted in increased expression of myeloid differentiation markers (CD33, CD11b and CD14). Here, we analyzed the molecular effect of AZA+VEN treatment in hematopoietic cell lines and higher-risk MDS patients, focusing on cell cycle, the expression of PLCs and myeloid differentiation markers.

Patients and Methods. THP-1 monocytic and MOLM-13 leukemic cells were used to test AZA 5 microM for 24h and 48h, alone or in combination with VEN 1 nM on cell cycle and the expression of PLCs and myeloid differentiation genes. MDS patients were 15 higher-risk subjects who had given informed consent according to the Declaration of Helsinki. Twelve patients were treated only with AZA (75 mg/m2/day for 5 days) and three cases were treated with both AZA and VEN (400 mg oral tablet once daily on days 1-14). All samples came from the Institute of Hematology "L e A Seràgnoli", Bologna, Italy, where also all clinical evaluations were made. MDS samples were collected at baseline and during therapy: the whole blood was used to perform a flow cytometric profiling, while mononuclear cells were used to carry out gene expression analyses. Real-time PCR quantified PLCbeta1, PLCgamma2, CD33, CD11b and CD14 expression, while GAPDH was the endogenous control and baseline levels within the same patient were used as the internal reference.

Results. In MOLM-13 cells, AZA+VEN significantly increased the sub-G0/G1 phase after only 24h (12.5% vs 1.5% control) and, even more, after 48 h (51.5% vs 1.6% control), as compared to AZA alone (5.3% at 24h and 29.2% at 48h). Even THP-1 cells showed an increased sub-G0/G1 phase after 24h (11.3% vs 1.8% control) or 48h (32.1% vs 2.9% control) of AZA+VEN, as compared with AZA alone (2.4% at 24h and 18% at 48h). At 24h, AZA+VEN THP-1 cells also showed an increased percentage of G0/G1 cells (58.0%), as compared with control (50.4%) and AZA alone (48.6%). Clinically, 7/12 patients treated with AZA alone were responders, achieving either hematologic improvement or complete remission. Moreover, they showed a higher PLCbeta1 and PLCgamma2 expression during treatment, as well as an increased expression of the myeloid differentiation markers. In contrast, the remaining 5/12 patients treated with AZA alone were non responders, showing a stable disease, and exhibited a reduction of PLCbeta1, PLCgamma2, CD33, CD11b and CD14. All 3 patients treated with AZA+VEN showed a rapid hematologic response, within the first three cycles of therapy (i.e., earlier than the median time to response in patients treated with AZA alone) and displayed a higher amount of both PLCs and the myeloid differentiation markers within the first two cycles of therapy. Moreover, flow cytometry analyses on MDS samples’ whole blood showed a reduced percentage of granulocytes during AZA+VEN therapy, as compared with responder patients treated with AZA alone.

Conclusions. The addition of VEN to AZA strongly and rapidly increases in vitro cell death and affects cell cycle. Similarly, the flow cytometric profiling of MDS cells during AZA+VEN administration shows a reduction of granulocytes that might be correlated with the myelotoxicity observed at a clinical level. Even PLCbeta1 and PLCgamma2, also related with G0/G1 regulation and myeloid differentiation, are early increased during AZA+VEN exposure, in both cell lines and MDS samples. In MDS, this increased expression is detectable from the very first cycles, even before the clinical response, that is very rapid with AZA+VEN therapy. All in all, our results may pave the way to a role for PLC molecular analyses in AZA+VEN response that must be confirmed in a larger number of patients. Further studies may also focus more on the BCL-2 inhibitory effect of VEN, that could regulate other PLC-related downstream molecules, possibly leading to new targeted therapies or molecular markers.

Cavo:AbbVie, Amgen, Bristol Myers Squibb/Celgene, Pfizer, GlaxoSmithKline, Sanofi, Roche, Takeda: Consultancy, Honoraria; Janssen: Honoraria, Speakers Bureau. Curti:Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Finelli:Takeda: Consultancy.