Investigating Phosphoinositide-Related and Apoptotic Pathways in Azacitidine and Venetoclax Combination Therapy in Higher-Risk Myelodysplastic Neoplasms and Leukemic Cell Lines
Authors: Casalin Irene , Ceneri Eleonora , De Stefano Alessia , Cappellini Alessandra , Pellagatti Andrea , Boultwood Jacqueline , Cavo Michele , Ratti Stefano , Manzoli Lucia , Cocco Lucio , Ferrari Anna , Simonetti Giorgia , Martinelli Giovanni , Fogli Miriam , Paolini Stefania , Parisi Sarah , Curti Antonio , Finelli Carlo , Follo Matilde Yung 
Published: December 2024

Background and Rationale. Azacitidine (AZA) is a key treatment for higher-risk Myelodysplastic Neoplasms (MDS). Adding Venetoclax (VEN) to AZA improves efficacy in Acute Myeloid Leukemia (AML) and higher-risk MDS (Bazinet and Bravo, 2022). Phospholipases C (PLCs) have been identified as important response biomarkers in higher-risk MDS (Cocco L et al, 2015), also due to their role in modulating apoptotic pathways through BCL-2 (Mongiorgi S et al, 2023). This study aims to explore the molecular effects of AZA and AZA+VEN on phosphoinositide-related and apoptotic pathways in higher-risk MDS patients, as well as in VEN resistant and sensitive leukemic cell lines.

Patients and Methods. 10 patients with higher-risk MDS (IPSS-R intermediate, high, or very high) (Greenberg et al, 2012) were recruited at the IRCCS-Institute of Hematology “L e A Seràgnoli”, Bologna, where all clinical evaluations were conducted. Samples were collected at baseline and during therapy, according to the Declaration of Helsinki and response to therapy was evaluated according to the IWG criteria (Cheson et al., 2006). 6/10 patients received AZA alone (75 mg/m2/day for 7 days), while 4 cases received AZA combined with VEN (400 mg/day oral tablet once daily on days 1-14). Mononuclear cells (MNCs) from peripheral blood or bone marrow were analyzed for baseline mutations and expression of PLCs and apoptotic markers.

THP-1 and MV4-11 leukemic cells, used as VEN resistant and sensitive in vitro models, were treated with VEN (respectively 125nM and 5nM) and AZA (5µM), either alone or in combination. After 24h cytofluorimetric, Real-Time PCR, Western blotting and immunofluorescence (IF) analyses were performed.

Results. A new BCL-2 frameshift mutation (p.Ala82Glyfs*14), possibly leading to a truncated protein, was found in 7/10 patients at baseline. Although its role is still unclear, preliminary results showed no correlation with treatment type or response. Instead, Responders (Complete remission (CR) or Haematological Improvement (HI)) to AZA alone showed reduced BCL-2 and increased BAX levels within the first 6 cycles, while Non-Responders (Stable Disease (SD)) exhibited the opposite pattern. All patients treated with AZA+VEN showed a rapid hematological response (CR), with early upregulation of both BCL-2 and BAX, followed by decreased expression in later cycles. Notably, one Responder (CR) patient treated with AZA+VEN, who later progressed to AML, showed increased BCL-2 expression shortly before AML evolution, with no significant changes in BAX levels.

After in vitro treatments, flow cytometry analyses showed a significant increase in Sub-G0 phase with AZA+VEN, while Annexin V assays and increased pro-apoptotic markers (BIM, BAK1) in both cell lines confirmed apoptosis. Treatment affected myeloid markers expression with an increase of CD11 and CD14 in both cell lines, while protein expression of PLCγ2 increased in THP-1 but decreased in MV4-11 cells. As for protein expression, pAKT (Ser473) and PP2A levels decreased in both cell lines, whereas pAMPKα decreased only in MV4-11 and pPKCα increased in THP-1 cells. Additionally, pGSK3, specifically the pGSK3β isoform, was phosphorylated in MV4-11 cells but was completely absent in THP-1 cells.

Conclusions. MDS patients responding to AZA+VEN showed altered BAX and BCL-2 expression, possibly increasing apoptosis. Moreover, MDS samples showed a novel BCL-2 frameshift mutation, whose clinical significance remains unclear. On the other hand, in vitro analyses on leukemic cell lines revealed altered cell cycle and phosphoinositide-dependent signaling pathways. These findings, warranting further research in a larger cohort of patients and additional leukemic cell lines, may contribute to a better understanding of the molecular mechanisms underlying the AZA and VEN combination therapy.