Systematic Evaluation of Preclinical Genetically Determined Vs. "Stochastic"/Transient Resistance to High-Dose Melphalan Treatment in Multiple Myeloma
Authors: Hengeveld Paul J. , Matthews Geoffrey M. , Hu Yiguo , Sheffer Michal , Bariteau Megan A. , Poarch Haley I. , Dhimolea Eugen , Mitsiades Constantine S 
Published: October 2019

Abstract
Myeloablative high-dose melphalan (HDM) followed by autologous stem cell transplantation (ASCT) remains one of the cornerstones of multiple myeloma (MM) treatment. Conceptually, HDM is based on the notion that a single melphalan dose (typically 200 mg/m2) exceeding the myelotoxicity threshold is associated with a steep increase in the dose response curve and higher degree of MM cell killing compared to fractionated delivery (without the need for hematopoietic stem cell support) of the same cumulative dose. Despite its importance in the therapeutic management of MM, HDM-ASCT is not considered a curative procedure, presumably because chemoresistant subpopulations of MM cells survive HDM and lead to eventual relapse. Elucidating the molecular mechanisms responsible for resistance to HDM in MM could have major implications for the identification of patients with the highest probability of major clinical benefit from this procedure. However, these resistance mechanisms remain incompletely understood. To address this void in the field, we examined whether an open-ended unbiased genome-wide functional characterization of MM cells could uncover genes associated with resistance to HDM and quantify the degree to which other, non-genetically determined, mechanisms of resistance contribute to this process. Specifically, we used the human myeloma cell line MM1.S, transduced with lentiviral construct for the Cas9 nuclease and with pooled lentiviral particles of the GeCKO library (Shalem et al., 2014), which consists of 2 pooled single guide RNA (sgRNA) sub-libraries (~120,000 sgRNAs; targeting ~19,000 genes and ~1800 miRNAs). Using this CRISPR/Cas9-based approach to mutagenize and cause loss of function of the genes recognized by the respective sgRNAs, we sought to facilitate the prospective isolation of MM cells resistant to HDM. To better simulate the exposure of MM cells to HDM in the autologous transplant setting, we adapted our in vitro treatment to include an initial dose of 25 µM of melphalan and sequential one-hour interval partial wash-outs, to achieve gradual reduction of melphalan concentrations, consistent with the pharmacokinetic profile observed in the clinical setting (Nath et al., 2010). We repeated twice the genome-wide knock-out screens, with biological replicates on both the transduction and cell culture level (maintaining a coverage of at least 1000 cells per individual sgRNA). While the in vitro simulation of clinical HDM with our wash-out regimen achieved >99% estimated reduction in MM cell viability in both screens, viable cells re-emerged in cultures from all biological and technical replicates. HDM-resistant cells were processed to quantify their sgRNA enrichment or depletion, using next generation sequencing, and also characterize the sensitivity of these MM cells to repeat exposure to HDM concentrations consistent with the genome-wide CRISPR screens. We observed that, despite having survived a previous round of exposure to HDM, the majority (>90%) of these sgRNA-transduced cells were again highly responsive to repeat treatment with HDM. A similar result was also obtained with MM1.S cells which had not been previously transduced with the sgRNA sub-libraries. While ongoing investigation in our lab addresses these findings on additional MM cell lines, these observations on a well-established MM cell line model of melphalan responsiveness raises the intriguing possibility that genetically-determined forms of HDM resistance, conferred after an unbiased genome scale evaluation through the CRISPR/Cas9-editing methodology, may only represent a small fraction of the MM cells which survive a round of HDM treatment, and that other non-genetically determined mechanisms may mediate the principal mode of resistance, even in the cell autonomous context of our experiments. In turn, this suggests that future efforts to individualize the administration of HDM-ASCT in patients and their post-ASCT monitoring should not rely exclusively on genetic markers identified and validated from preclinical experimentation with repeated rounds of MM cell exposure to the equivalent of myelotoxic melphalan concentrations. Instead, renewed emphasis is warranted on identification of functional markers correlating with the ability of MM cells to develop transient resistance to HDM, even in the absence of constitutive genetically-determined mechanisms of resistance.

Disclosures
Mitsiades: Novartis: Research Funding; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding.