Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. .

28The myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells 29 and resistance to therapy. Interleukin-6 (IL-6) and downstream JAK/STAT signaling are thought 30 to be central components of these microenvironment-induced phenotypes. In a prior drug 31 repurposing screen, we identified tofacitinib, a pan-JAK inhibitor FDA-approved for rheumatoid 32 arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow 33 mesenchymal stromal cells. Here, we validated both in vitro, in stromal-responsive human 34 myeloma cell lines, and in vivo, in orthotopic disseminated murine xenograft models of 35 myeloma, that tofacitinib showed both single-agent and combination therapeutic efficacy in 36 myeloma models. Surprisingly, we found that ruxolitinib, an FDA-approved agent targeting 37 JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel 38 irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. RNA-seq and 39 unbiased phosphoproteomics revealed that marrow stromal cells stimulate a JAK/STAT-40 mediated proliferative program in myeloma plasma cells, and tofacitinib reversed the large 41 majority of these pro-growth signals. Taken together, our results suggest that tofacitinib 42 specifically reverses the growth-promoting effects of the tumor microenvironment through 43blocking an IL-6-mediated signaling axis. As tofacitinib is already FDA-approved, these results 44 can be rapidly translated into potential clinical benefits for myeloma patients. 45 46 47 . CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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AbstractEnhancing the efficacy of proteasome inhibitors is a central goal in myeloma therapy. We proposed that signaling-level responses after PI would reveal new mechanisms of action that could be therapeutically exploited. Unbiased phosphoproteomics after the PI carfilzomib surprisingly demonstrated the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation was invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrated broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings led us to evaluate direct spliceosome inhibition in myeloma, which synergized with carfilzomib and showed potent anti-tumor activity. Functional genomics and exome sequencing further supported the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.SignificanceNew ways to enhance PI efficacy are of major interest. We combine systems-level analyses to discover that PIs specifically interfere with splicing and that myeloma is selectively vulnerable to spliceosome inhibition. We reveal a new approach to advance myeloma therapy and uncover broader roles of splicing modulation in cancer.