Loss of GABARAP mediates resistance to immunogenic chemotherapy in multiple myeloma.

Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant anti-tumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABARAP is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in high-risk MM patients. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent anti-tumor T cell response. Low GABARAP was independently associated with shorter MM patient survival and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, like bortezomib, with an autophagy inducer, like rapamycin, may improve patient outcomes in MM, where low GABARAP in the form of del(17p) is common and leads to worse outcomes.

Ubiquitin receptor PSMD4/Rpn10 is a novel therapeutic target in multiple myeloma.

PSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Because proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells compared with normal plasma cells. Rpn10 levels inversely correlated with overall survival in patients with MM. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with the activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation, and the activation of CD4+ T and natural killer cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary cells from patients with MM with SB decreased cell viability without affecting the viability of normal peripheral blood mononuclear cells. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI) resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well tolerated, inhibited tumor growth, and prolonged survival. Our data suggest that inhibiting Rpn10 will enhance cytotoxicity and overcome PI resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.

A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

Identification and validation of ecto-5' nucleotidase as an immunotherapeutic target in multiple myeloma.

Interaction of plasmacytoid dendritic cells (pDCs) with multiple myeloma (MM) cells, T- or NK-effector cells in the bone marrow (BM) microenvironment induces tumor cell growth, as well as inhibits innate and adaptive immune responses. Defining pDC-MM interaction-triggered immunosuppressive mechanism(s) will enable design of interventional therapies to augment anti-MM immunity. In the present study, we show that pDC-MM interactions induce metabolic enzyme Ecto-5' Nucleotidase/CD73 in both pDCs and MM cells. Gene expression database from MM patients showed that CD73 levels inversely correlate with overall survival. Using our pDC-MM coculture models, we found that blockade of CD73 with anti-CD73 Abs: decreases adenosine levels; activates MM patient pDCs; triggers cytotoxic T lymphocytes (CTL) activity against autologous patient MM cells. Combination of anti-CD73 Abs and an immune-stimulating agent TLR-7 agonist enhances autologous MM-specific CD8+ CTL activity. Taken together, our preclinical data suggest that the therapeutic targeting of CD73, alone or in combination with TLR-7 agonist, represents a promising novel strategy to restore host anti-MM immunity.

A 3D-Bioprinted Multiple Myeloma Model.

Multiple myeloma (MM) is a malignancy of plasma cells accounting for ≈12% of hematological malignancies. In this study, the fabrication of a high-content in vitro MM model using a coaxial extrusion bioprinting method is reported, allowing formation of a human bone marrow-like microenvironment featuring an outer mineral-containing sheath and the inner soft hydrogel-based core. MM cells are mono-cultured or co-cultured with HS5 stromal cells that can release interleukin-6 (IL-6), where the cells show superior behaviors and responses to bortezomib in 3D models than in the planar cultures. Tocilizumab, a recombinant humanized anti-IL-6 receptor (IL-6R), is investigated for its efficacy to enhance the chemosensitivity of bortezomib on MM cells cultured in the 3D model by inhibiting IL-6R. More excitingly, in a proof-of-concept demonstration, it is revealed that patient-derived MM cells can be maintained in 3D-bioprinted microenvironment with decent viability for up to 7 days evaluated, whereas they completely die off in planar culture as soon as 5 days. In conclusion, a 3D-bioprinted MM model is fabricated to emulate some characteristics of the human bone marrow to promote growth and proliferation of the encapsulated MM cells, providing new insights for MM modeling, drug development, and personalized therapy in the future.

Bortezomib induces anti-multiple myeloma immune response mediated by cGAS/STING pathway activation.

Proteasome inhibitor bortezomib induces apoptosis in multiple myeloma (MM) cells, and has transformed patient outcome. Using in vitro as well as in vivo immunodeficient and immunocompetent murine MM models, we here show that bortezomib also triggers immunogenic cell death (ICD) characterized by exposure of calreticulin on dying MM cells, phagocytosis of tumor cells by dendritic cells, and induction of MM specific immunity. We identify a bortezomib-triggered specific ICD-gene signature associated with better outcome in two independent MM patient cohorts. Importantly, bortezomib stimulates MM cells immunogenicity via activation of cGAS/STING pathway and production of type-I interferons; and STING agonists significantly potentiate bortezomib-induced ICD. Our studies therefore delineate mechanisms whereby bortezomib exerts immunotherapeutic activity, and provide the framework for clinical trials of STING agonists with bortezomib to induce potent tumor-specific immunity and improve patient outcome in MM.

Proteomic analysis identifies mechanism(s) of overcoming bortezomib resistance via targeting ubiquitin receptor Rpn13.

Our prior study showed that inhibition of 19S proteasome-associated ubiquitin receptor Rpn13 can overcome bortezomib resistance in MM cells. Here, we performed proteomic analysis of Rpn13 inhibitor (RA190)-treated MM cells and identified an antioxidant enzyme superoxide dismutase (SOD1) as a mediator of Rpn13 signaling. SOD1 levels are higher in MM patient cells versus normal PBMCs; and importantly, SOD1 expression correlates with the progression of disease and shorter survival. Functional validation studies show that RA190-induced cytotoxicity in bortezomib-sensitive and -resistant MM cells is associated with decrease in SOD1 levels; conversely, forced expression of SOD1 inhibits RA190-induced cell death. Genetic knockdown and biochemical blockade of SOD1 with LCS-1 sensitizes bortezomib-resistant MM cells to bortezomib. SOD1 inhibitor LCS-1 decreases viability in MM cell lines and patient cells. LCS-1-induced cell death is associated with: (1) increase in superoxide and ROS levels; (2) activation of caspases, and p53/p21 signaling; (3) decrease in MCL-1, BCLxL, CDC2, cyclin-B1, and c-Myc; (4) ER stress response; and (5) inhibition of proteasome function. In animal model studies, LCS-1 inhibits xenografted bortezomib-resistant human MM cell growth and prolongs host survival. Our studies therefore show that targeting Rpn13 overcomes bortezomib resistance by decreasing cellular SOD1 levels, and provide the rationale for novel therapeutics targeting SOD1 to improve patient outcome in MM.

The E3 ligase HUWE1 inhibition as a therapeutic strategy to target MYC in multiple myeloma.

Proteasome inhibitors have provided a significant advance in the treatment of multiple myeloma (MM). Consequently, there is increasing interest in developing strategies to target E3 ligases, de-ubiquitinases, and/or ubiquitin receptors within the ubiquitin proteasome pathway, with an aim to achieve more specificity and reduced side-effects. Previous studies have shown a role for the E3 ligase HUWE1 in modulating c-MYC, an oncogene frequently dysregulated in MM. Here we investigated HUWE1 in MM. We identified elevated expression of HUWE1 in MM compared with normal cells. Small molecule-mediated inhibition of HUWE1 resulted in growth arrest of MM cell lines without significantly effecting the growth of normal bone marrow cells, suggesting a favorable therapeutic index. Studies using a HUWE1 knockdown model showed similar growth inhibition. HUWE1 expression positively correlated with MYC expression in MM bone marrow cells and correspondingly, genetic knockdown and biochemical inhibition of HUWE1 reduced MYC expression in MM cell lines. Proteomic identification of HUWE1 substrates revealed a strong association of HUWE1 with metabolic processes in MM cells. Intracellular glutamine levels are decreased in the absence of HUWE1 and may contribute to MYC degradation. Finally, HUWE1 depletion in combination with lenalidomide resulted in synergistic anti-MM activity in both in vitro and in vivo models. Taken together, our data demonstrate an important role of HUWE1 in MM cell growth and provides preclinical rationale for therapeutic strategies targeting HUWE1 in MM.

Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism.

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.

Melflufen plus dexamethasone in relapsed and refractory multiple myeloma (O-12-M1): a multicentre, international, open-label, phase 1-2 study.

BACKGROUND: Multiple myeloma is an incurable haematological malignancy, representing over 10% of haematological cancers in the USA. We did a phase 1-2 study of melflufen and dexamethasone in patients with relapsed and refractory multiple myeloma to determine the maximum tolerated dose of melflufen and to investigate its safety and efficacy. METHODS: We did a multicentre, international, dose-confirmation and dose-expansion, open-label, phase 1-2 study in seven centres in the USA and Europe. Eligible patients were aged 18 years or older, had relapsed and refractory multiple myeloma, had received two or more previous lines of therapy (including lenalidomide and bortezomib), were refractory to their last line of therapy, and had an Eastern Cooperative Oncology Group performance status of 2 or less. In phase 1, patients received an intravenous infusion of melflufen at 15 mg, 25 mg, 40 mg, or 55 mg for 30 min on day 1 in 21-day cycles plus oral dexamethasone 40 mg weekly and did not receive melflufen as a single agent. Melflufen was also tested in a single-agent cohort late in phase 2 in a small number of patients at the maximum tolerated dose identified in phase 1. In phase 2, patients were enrolled at the maximum tolerated dose in the melflufen plus dexamethasone in the combination cohort.. The phase 1 primary objective was to determine the maximum tolerated dose. The phase 2 primary objective was to evaluate overall response rate and clinical benefit rate. This primary analysis was done per protocol, in the all-treated and efficacy-evaluable population (defined as patients who received at least two doses of melflufen and who had a response assessment after baseline). The single-agent melflufen cohort was closed on October 6, 2016, as per the recommendation by the data safety monitoring committee on the basis of interim data suggesting greater activity in the melflufen plus dexamethasone cohort. The study is completed but survival follow-up is ongoing. This study is registered with ClinicalTrials.gov, NCT01897714. FINDINGS: Patients were enrolled between July 4, 2013, and Dec 31, 2016: 23 patients in phase 1 and 58 in phase 2, including six patients from phase 1 treated at the maximum tolerated dose of melflufen 40 mg plus weekly dexamethasone. In phase 2, 45 patients were given a combination of melflufen plus dexamethasone and 13 patients were given single-agent melflufen. In phase 1, the established maximum tolerated dose was 40 mg of melflufen in combination with dexamethasone. No dose-limiting toxicities were observed in the first three dose cohorts (15 mg, 25 mg, and 40 mg). The highest dose cohort tested (55 mg) exceeded the maximum tolerated dose because four of six patients experienced grade 4 neutropenia with grade 4 thrombocytopenia also occurring in three of these patients; therefore, the planned highest dose of 70 mg was not tested. In phase 2, patients treated with combination therapy achieved an overall response rate of 31% (14 of 45 patients; 95% CI 18-47) and clinical benefit rate of 49% (22 of 45; 34-64) in the all-treated population, and 41% (14 of 34; 25-59) and 65% (22 of 34; 47-80) in the efficacy-evaluable population. In the phase 2 single-agent cohort, the overall response rate was 8% (one of 13 patients; 0·2-36·0) and the clinical benefit rate was 23% (three of 13; 5-54). Among the 45 patients given melflufen plus dexamethasone during phase 2, the most common grade 3-4 adverse events were clinically manageable thrombocytopenia (28 [62%] patients) and neutropenia (26 [58%]), and non-haematological toxicity was infrequent. 24 serious adverse events were reported in 17 (38%) of 45 patients, most commonly pneumonia (five [11%]). The most common grade 3-4 adverse events that occurred in the phase 2 single-agent cohort of 13 patients were neutropenia (nine [69%]) and thrombocytopenia (eight [62%]). Nine patients experienced serious adverse events in the single-agent cohort, most commonly thrombocytopenia (two [15%]). There were three deaths from adverse events within 30 days of treatment that were possibly related to treatment: one in the 25 mg cohort in phase 1 (due to bacteraemia) and two in the phase 2 combination cohort (one due to neutropenic sepsis and one due to Escherichia coli sepsis), each in the setting of progressive disease. INTERPRETATION: These data show that melflufen is active in patients with relapsed and refractory multiple myeloma and tolerable in most patients. These results show the feasibility of this regimen and support the initiation of additional clinical studies of melflufen in multiple myeloma, both in combination with dexamethasone as well as in triplet regimens with additional classes of drugs. FUNDING: Oncopeptides AB.

Preclinical validation of Alpha-Enolase (ENO1) as a novel immunometabolic target in multiple myeloma.

Bone marrow plasmacytoid dendritic cells (pDCs) in patients with multiple myeloma (MM) promote tumor growth, survival, drug resistance, and immune suppression. Understanding the molecular signaling crosstalk among the tumor cells, pDCs and immune cells will identify novel therapeutic approaches to enhance anti-MM immunity. Using oligonucleotide arrays, we found that pDC-MM interactions induce metabolic enzyme Alpha-Enolase (ENO1) in both pDCs and MM cells. Analysis of MM patient gene expression profiling database showed that ENO1 expression inversely correlates with overall survival. Protein expression analysis showed that ENO1 is expressed in pDC and MM cells; and importantly, that pDC-MM coculture further increases ENO1 expression in both MM cells and pDCs. Using our coculture models of patient autologous pDC-T-NK-MM cells, we examined whether targeting ENO1 can enhance anti-MM immunity. Biochemical inhibition of ENO1 with ENO1 inhibitor (ENO1i) activates pDCs, as well as increases pDC-induced MM-specific CD8+ CTL and NK cell activity against autologous tumor cells. Combination of ENO1i and anti-PD-L1 Ab or HDAC6i ACY-241 enhances autologous MM-specific CD8+ CTL activity. Our preclinical data therefore provide the basis for novel immune-based therapeutic approaches targeting ENO1, alone or in combination with anti-PD-L1 Ab or ACY241, to restore anti-MM immunity, enhance MM cytotoxicity, and improve patient outcome.