Heightened Endoplasmic-Reticulum Stress Is a Targetable Vulnerability of Malignant Plasma Cells in Multiple Myeloma

Background: In multiple myeloma (MM), malignant plasma cells (PC) secret a high load of non-functional monoclonal immunoglobulins and cytokines. These non-functional proteins contain high levels of disulfide bonds, crucial for proper folding and structural integrity to avoid the unfolded protein response (UPR)-induced apoptosis and clonal extinction. Protein disulfide isomerase A1 (PDIA1) is the only known ER-resident enzyme isoform to meet this need by connecting intramolecular cysteine residues. Heightened endoplasmic reticulum (ER) function to maintain proteostasis in the context of enhanced secretory burden is the hallmark of malignant PC; hence perturbation of proteostasis in the ER can result in vulnerability and apoptosis of PC. To date, disruption of proteostasis has proven to be overwhelmingly efficacious in MM with the use of proteasome inhibitors (PI), however, resistance to PI therapy emerges in most cases, emphasizing the need for new therapies. Inhibiting the PDIA1 function may constitute a novel alternative method to target proteostasis due to the aforementioned reliance of malignant PC. This may provide a therapeutic window to preferentially induce lethality to MM cells compared to normal bone marrow cells which are less dependent on amplified ER function. Earlier we reported a small "lead” compound highly specific to PDIA1. Here we demonstrate that PI resistant MM cell lines and primary patient-derived MM cells have high sensitivity to the next generation of PDIA1 inhibitors.

Methods/Results: To further clarify determinants of PI and PDIA1 inhibitor sensitivity, we analyzed gene expression patterns in CD138⁺ MM cells via RNA sequencing. Consistent with our previous findings, expression of PDIA1 was significantly higher in patients with early and late relapsed MM, suggesting that PDIA1 may play a critical role in the PI resistant/relapsed state. This elevated PDIA1 expression in PC could be a resistance factor. Therefore, we hypothesized that PDIA1 is a marker for sensitivity of MM cells, thus PD1A1 inhibition may allow for a targeted application of this class of agents to overcome PI resistance.

Earlier, using high throughput screening coupled with structure-based medicinal chemistry approach, we developed and optimized a highly PDIA1 specific inhibitor CCF642-34. In a pre-clinical model of different PC lines made in our laboratory, inhibition of PDIA1 using CCF642-34 was highly effective, with bortezomib (BTZ) resistant MM cells being 2X more sensitive compared to BTZ naïve cells. We concluded that this sensitivity gradient is due to increased expression of PDIA1 as necessary compensatory mechanism to maintain proteostasis. Moreover, chemical inhibition of PDIA1 was also highly effective against BTZ resistant patient-derived MM cells whereby CCF642-34 has shown a nearly 4-fold superior potency in restricting the MM cells in vitro cell culture conditions. Interestingly, the same concentration of PDIA1 has no cytotoxic effect on the CD34⁺ cells derived from healthy bone marrow. Our data show that the new generation of PDIA1 inhibitors induced unresolvable ER stress in MM cells as observed in a dose- and time-dependent expression of CHOP transcription factor, ultimately resulting in profound induction of apoptosis as measured by PARP and caspase 3 cleavage. Consistent with our earlier reports, analysis of RNA NGS results of primary MM cells treated with PDIA1 inhibitor demonstrated its selectivity in inducing ER-stress, particularly UPR, ER-associated protein degradation (ERAD) and ATF6/PERK pathway. Most interestingly, CCF642-34 was highly effective in restricting the emergence of disease in syngeneic in vivo murine models of Vk*MYC transgenic transplant models of MM. We found that treatment of Black6 mice (n= 6/gp, 10mg/Kg, 3 days/wk p.o) inoculated with Vk*MYC MM cells gave significant survival benefit compared to vehicle control.

Conclusion: Here we demonstrate that the resistance to PI in patients with MM occurs, in part, due to the gain in the ER function, primarily via upregulation of PDIA1, an ER resident disulfide isomerase, critical for protein folding. The increased dependence of MM cells on ER function makes them highly vulnerable to PDIA1 disruption, particularly upon PI resistance acquisition. We have developed a next generation orally bioavailable PDIA1 inhibitor that demonstrated high therapeutic index in pre-clinical model systems.

Shain:Bristol Myers Squibb (BMS), Janssen, GlaxoSmithKline (GSK), Adaptive, Sanofi, and Takeda, and Amgen: Honoraria; GSK, Janssen and BMS: Membership on an entity's Board of Directors or advisory committees; GSK, BMS, Sanofi, Karyopharm, Takeda, Janssen, Adaptive and Amgen: Speakers Bureau; AbbVie and Karyopharm: Research Funding; Janssen and BMS: Other: PI of clinical trials. Maciejewski:Alexion: Consultancy; Apellis Pharmaceuticals: Consultancy.