Role of phosphoinositide-dependent signaling and optimization of PDZD2 genome editing in vitro model in myelodysplastic neoplasms and acute myeloid leukemia

Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML) are hematologic malignancies marked by ineffective hematopoiesis, differentiation defects, and profound epigenetic dysregulation.These alterations drive disease heterogeneity and therapeutic resistance, emphasizing the need for mechanistic insights to guide new treatments. In the first part of this study, we investigated the molecular effects of Azacitidine (AZA) and Venetoclax (VEN), alone and in combination, in high-risk MDS (HR-MDS) patients and AML cell lines. We identified a recurrent BCL2 frameshift mutation at baseline, which may influence sensitivity to AZA+VEN through non-canonical mechanisms. Analyses of patient-derived hematopoietic cells suggested interplay between phosphoinositide-dependent signaling and apoptotic pathways, further confirmed in leukemic cell lines. In these models, AZA+VEN induced caspase-dependent apoptosis, modulated BCL2 family proteins, and altered PLC/PKC and AKT/GSK3 signaling pathways, providing novel insight into the molecular basis of response and resistance to this combination therapy. The second part of the project focused on developing a CRISPR-Cas9 genome-editing platform to investigate PDZD2, a poorly characterized gene hypermethylated in nearly 90% of AML cases and proposed as an epigenetic regulator of hematopoiesis. Conducted at Dr. Figueroa’s laboratory at the University of Miami, this work established targeted editing in HEK293 cells, introducing defined modifications at the PDZD2 locus—including an HA tag, a CASP3 cleavage-site mutation, and NLS alterations. Sequencing analyses revealed locus-dependent variability, with efficient homozygous knock-ins at the CASP3 site but lower HDR rates and instability for the HA and NLS modifications. Together, these two complementary projects integrate translational and mechanistic perspectives. The AZA+VEN study elucidates adaptive signaling networks with direct therapeutic relevance, while the PDZD2 genome-editing platform provides a methodological foundation to dissect epigenetic regulators in hematopoiesis. Collectively, this work advances our understanding of the molecular and epigenetic mechanisms driving leukemic progression and treatment response.