Auburn Research Day 2022

Sayak Chakravarti* 1 ; Harish Kumar 1 ; Suman Mazumder 1, 2 ; Sabyasachi Sanyal 3 ; Brian G. Van Ness 4 ; Amit Kumar Mitra 1, 2 1 Harrison School of Pharmacy, Auburn University, Auburn, AL; 2 Center for Pharmacogenomics and Single-cell Omics Initiative, Auburn University, Auburn, AL; 3 CSIR-Central Drug Research Institute, Lucknow, India; 4 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN B i omed i ca l Resea rch | Gr adua te/Undergr adua te St udent The Anti-Leprosy Drug Clofazimine Synergizes with BTK Inhibitors and Proteasome Inhibitors in Drug-Resistant Mantle Cell Lymphoma 65

Mantle Cell Lymphoma (MCL) is a difficult to cure, highly heterogeneous, & aggressive form of non-Hodgkin lymphoma with high recurrence rate & poor long-term prognosis (reported progression-free survival of 1-2 years). Although patients respond well initially to current first-line MCL therapies that includes combination regimens like R-CHOP, most eventually progress to relapsed disease state. BTKis (Bruton’s tyrosine kinase inhibitor/ Ibrutinib) and proteasome inhibitor (PI) drug Bortezomib/Bz are standard targeted therapeutic options for refractory/ relapsed (R/R) MCL. However, despite of recent advances in treatment landscape, R/R MCL still remains incurable with limited therapeutic options & median OS <10- 15 months. Therefore, there is an unmet need to discover novel drugs against R/R MCL. Previously, we have demonstrated Clofazimine (CLF), an anti-leprosy drug, could potentially be repurposed for the treatment of chronic myeloid leukemia where it targeted the subclones representing putative stem-like-cells (CSCs). Using single-cell analysis & high dimensional immunophenotyping, we also identified molecular networks underpinning CLF+PI synergy. We hypothesize that CLF has strong potential to be repurposed as novel anti MCL drug, particularly in relapsed/refractory setting. For this purpose, we used MCL cell lines representing drug-sensitive (JEKO1, MINO), innate BTKi-resistant (Z138) & clonally derived acquired PI-resistant (MINO-R) as in vitro model systems and showed i) the efficacy of CLF as single agent (IC 50 = 6.9±3.6 uM) and ii) in combination with PIs & BTKis against innate & acquired resistant MCL as well as iii) the unique targeting of putative CSCs by CLF. Further, mRNA-sequencing followed by differential gene expression analysis revealed that top significantly upregulated genes following PI+CLF treatment were GSR, DAP3, and DOK1, with reported anti-tumorigenic activity. The top significantly downregulated genes EHD1, CBX8, DDX17, SOX12 have reported pro-survival function. Ingenuity pathway analysis revealed protein ubiquitination pathway & cell cycle arrest at the G2/M phase as top canonical pathways. PI+CLF combination potentiated AMPK

mediated down-regulation of the mTOR signaling pathway, which further led to direct reduction of Cyclin D1 (aberrantly expressed in MCL) & downregulation of eIF4-p70S6K signaling. Recent studies have indicated intra-tumor heterogeneity due to the presence of stem-like cells in MCL (MCL-CSCs), including CD45+CD19- MCL- initiating cells (MCL-ICs) may drive drug resistance & disease relapse. We found that several of the differentially regulated genes are critical for the maintenance & functioning of CSCs. For example, PI+CLF combination downregulates Wnt/ β catenin signaling & HIPPO signalling which are found to be frequently overexpressed in MCL-ICs. Currently, we are validating the specific targeting of putative MCL- CSCs by CLF. Our study, thus introduces CLF as a novel therapeutic option for the management of R/R MCL.

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