Validation of novel secondary therapies in relapsed/refractory cancers using Pharmacogenomics and Single-Cell transcriptomics
Type of DegreeMaster's Thesis
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Multiple myeloma is the second-most common hematopoietic malignancy in the United States. Although several drugs have so far been approved by the FDA to treat myeloma and other plasm cell neoplasms, myeloma remains an incurable disease with dose-limiting toxicities and resistance to primary drugs like proteasome inhibitors (PIs) and Immunomodulatory drugs (IMiDs). We have recently established a novel pipeline called secDrug that incorporates a pharmacogenomics data-driven computational algorithm to introduce several new secondary drug candidates (secDrugs) against PI and IMiD-resistant myeloma, with rank and confidence scores. Furthermore, the Ras is known to represent the most mutated oncogene in >20% of myeloma patients which is also associated with greater tumor burden and cancer aggressiveness. Therefore, the overall objective of this study is to validate the efficacy of the top-ranked predicted secDrug, EHT1864 - an inhibitor of Rac1, a GTPase from Ras superfamily, using myeloma cell lines mirroring the extensive inter-tumor and intra-tumor heterogeneity of relapsed and refractory myeloma patients. First, we used single-cell RNA sequencing (RNA-Seq) as a screening tool to demonstrate that EHT1864 is potentially effective against myeloma subclones based on the enrichment of target genes. For in vitro validation, we used a panel of human myeloma cell lines (HMCLs) representing drug-sensitive (FLAM76), and innate/refractory resistance (LP1). We acquired/relapsed resistance (parental and clonally derived PI-resistant and IMiD-resistant HMCL pair U266 P/VR, RPMI P/VR, MM1S P/LenR). Flam 76 K-Ras (FLAM76 K12) and Flam-76 Nras (FLAM76 N12) cell lines were generated using Adeno-associated viral (AAV) vector-mediated delivery of CRISPR-Cas9 for genome editing in humans. Ras mutations were validated using the Sanger DNA sequencing method. Cell viability was measured using Cell Titer Glo assay (which measures viability using ATP produced by living cells). Cell death due to apoptosis was investigated using a combination of flow cytometry (Annexin-PI assay), Caspase 3/7 activity assay, and immunoblotting (assessment of cleaved caspase-3 and cleaved caspase-9, and several apoptotic markers). Our results showed that EHT1864 not only shows substantial potency as single-agent in HMCLs (IC50 range – 15-40uM) but also synergizes with PIs (represented by Ixazomib) and IMiDs (represented by Lenalidomide). CI values were consistently <0.9, demonstrating high synergy. Furthermore, DRI >1 represented a significant decrease in the predicted effective dose of PIs when used as a combination. This alludes to a potential decrease in dose-limiting toxicities if EHT1864-containing combination regimens are introduced in clinical scenarios. Interestingly, EHT1864 was more potent against drug-resistant and Ras-mutant HMCLs. This is important since we also found that Ras mutations (KRas 12 and NRas 12) were associated with significantly lower PI response in our larger panel of myeloma lines. Further, we showed that EHT1864 treatment also affects cell migration and mitochondrial membrane potential. Currently, we are performing pre-vs-post-treatment single-cell RNA sequencing (RNA-Seq) analysis to evaluate the intra-tumor efficacy of EHT1864 based on changes in the subclonal landscape of subclonal clusters within myeloma tumors. In this ongoing research endeavor, next, we will perform genome-wide transcriptome analysis (bulk RNA sequencing) to derive signatures representing the basis on-target and off-target efficacy of EHT1864, EHT1864+PI, and EHT1864+IMiD therapy. Results will be confirmed using qPCR and Immunoblotting analysis. Finally, we will perform single-cell proteomics (CyTOF or Cytometry time of flight) in patient-derived CD138+ bone marrow cells (ex vivo) to establish the molecular pathways underlying secDrug efficacy and drug synergy. Our long-term goal is to perform CRISPR-based gene editing and functionally validate these mechanisms. Thus, our work lays a framework to establish a novel Rac1 inhibitor as a potent clinical trial-ready therapeutic option for the management of PI and IMiD-resistant myeloma.