Repurposing Clofazimine As a Novel Drug for the Treatment of PI-Resistant Stem Cell-like Subclones in Myeloma

Multiple myeloma (MM) is the second-most common hematopoietic malignancy in the United States with estimated new reported cases of 32,110 and deaths of 12,960 in 2019. Despite recent improvement in therapies, including proteasome inhibitors (PIs) - the standard-of-care drug, MM still remains a difficult-to-cure disease with significant complexity and heterogeneity at the molecular level. Recent studies have shown that the presence of cell sub populations with potential cancer stem cell-like properties (MM-CSCs), including CD19^- CD138^- quiescent stem cells, dormant cells, ALDH⁺ cells and side populations/SP, in drug-resistant tumors significantly contribute to the development of PI resistance in myeloma. However, no study so far has attempted to develop drugs specifically targeting MM-CSCs involved in drug resistance. Further, the gene signature underlying these sub cellular populations are yet to be revealed.

We have previously demonstrated that Clofazimine (CFZ), an anti-leprosy drug, could be successfully repurposed for the treatment of Chronic myeloid leukemia (CML) by specifically targeting quiescent stem cell population (CD34⁺CD38^-, CFSE-bright) in drug resistant patient samples. Here, we hypothesized that we will repurpose CFZ as a novel drug for the treatment of PI-resistant MM by targeting the sub-cellular stem cell-like subclones.

Therefore, in the current study we utilized our panel of >50 human myeloma cell lines (HMCLs) representing innate PI-response/resistance and >10 pairs (parental and PI-resistant lines generated using dose escalation over a period of time) of clonally derived PI-resistant HMCLs representing acquired resistance to repurpose CFZ for the management of PI-resistant MM. Cells were treated with CFZ and the PIs Bortezomib (Bz), Carfilzomib (Cz), Ixazomib (Ix) and Oprozomib (Opz) as single agents or in combination and in vitro cytotoxicity was determined using CellTiter-Glo assay. Synergy between CFZ and PIs was analyzed by Calcusyn software based on Chou-Talalay's combination index (CI) theorem. Drug-induced apoptosis, ROS generation, mitochondrial membrane potential and cell cycle arrest were evaluated by Flow cytometry. Sub-cellular stem cell-like populations were isolated using cell sorting by Flow Cytometry (FCM/FACS). Tumorigenic potential of purified MM-CSCs was determined using colony forming assay, carboxyfluorescein succinimidyl ester (CFSE) assay, Aldeflour activity and analysis of side population in resistant MM cells.

We found very promising preliminary results where CFZ alone showed very potent inhibition of cell viability in HMCLs. Further, treatment with CFZ + PIs significantly improved the therapeutic index of PI administration to the cells. Similar results were observed in parental (P) vs clonally derived PI-resistant (VR) cell lines. We next evaluated if CFZ alone or in combination with PIs could erode quiescent CD138⁺ cells in parental-P/clonally derived PI-resistant cells-VR. Hence, we labelled RPMI-8226 (parental cells), RPMI8226-VR with CFSE and treated them with drugs for 96h. While PIs failed to reduce CFSE-bright (non-dividing) cells, CFZ alone or in combination with Ixazomib significantly reduced their number and increased CFSE-dim (dividing cell) population. Evaluation of apoptosis in these cells revealed that CFZ alone caused apoptosis in both CFSE-bright and CFSE-dim cells while combining CFZ +PI caused a more robust effect amounting to their near-obliteration. We next assessed side populations/SP in resistant cells. We found that % SP was higher in resistant cells as compared to parental cells. CFZ alone or in combination (CFZ+PI) reduced %SP in PI- resistant cell lines.

Based on these results, we propose that CFZ may have strong potential to increase the therapeutic efficacy of PIs when used in combination by specifically targeting MM-CSCs sub cellular population in MM - which we are investigating further. Currently, we are: 1) validating the potency of CFZ (alone or CFZ+PI combination therapy) in primary myeloma cells/PMCs from newly diagnosed patients, followed by 2) bulk mRNA sequencing and 3) single-cell transcriptomic analysis of MM-CSCs in HMCLs and PMCs to evaluate molecular pathways involved in stem-cell based PI-resistance and to characterize PI-resistant sub-cellular stem cell populations based on gene expression signatures using our prediction analysis software SCATTome.