Bianchi, Giada

Abstract Introduction: The bone marrow (BM) niche promotes multiple myeloma (MM) growth, survival and drug resistance. Therapies targeting both cancer cells and the microenvironment are highly effective. We were interested in identifying novel signaling pathways supporting MM pathogenesis through MM-BM crosstalk. The transmembrane receptor Roundabout 1 (ROBO1) plays a role in growth and dissemination of solid tumors, however its function in MM is unknown. Material and Methods: We analyzed ROBO1 expression in cell lines and primary samples via western blot, immunohistochemistry (IHC) and gene expression profiling. We used short hairpin RNA and CRISPR-Cas9 for ROBO1 knock down (KD) and knock out (KO), respectively. For protein structure-function and rescue studies, we stably expressed full-length (FL) or mutant ROBO1 devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived G674* truncation, with a C-terminus FLAG tag. We used a 3D hydrogel encapsulation system to study proliferation; FLAG immunoprecipitation (IP) followed by mass spectrometry to identify ROBO1 interacting partners; and immunofluorescence to detect ROBO1 localization. To study tumor growth in vivo, we performed PET-CT of mice inoculated subcutaneously or intramedullary with WT or ROBO1 KO MM cells and retrieved tumors for RNA sequencing. To study dissemination and homing, KO and FL addback MM cells were injected intravenously in SCID mice. Mice were monitored for development of tumors or hindlimb paralysis and femora/tumors harvested once mice reached endpoint. Results: ROBO1 is highly expressed in MM cell lines and primary cells but low/absent in normal plasma cells and other hematologic cancer cell lines. ROBO1 KD is specifically cytotoxic for MM cells and ROBO1 KO decreases proliferation, a phenotype fully rescued by FL ROBO1. Compared to WT, ROBO1 KO significantly decreases intramedullary (mean tumor volume (MTV): 1323 vs 457 mm3, p value= 0.02) and extramedullary (MTV: 2684 vs 823 mm3, p value= 0.001) tumor growth in vivo. We further discovered that ROBO1 KO decreases adhesion of MM to BM endothelial and BMSC, which is fully rescued by FL ROBO1. In a disseminated mouse model, ROBO1 KO cells generate bone plasmacytoma with reduced BM invasion, as compared to the extensive BM infiltration observed with ROBO1 FL cells. Consistently, in primary samples from patients, we detected ROBO1 expression only in 1 out of 10 solitary plasmacytoma (dim staining) as compared to 14 out of 14 MM bone marrow biopsy samples tested (11 strong, 3 dim, p value= 0.0001). Mechanistically, we show for the first time that ROBO1 C-terminus is cleaved in a ligand-independent fashion; translocates to the nucleus; and is necessary and sufficient to rescue ROBO1 KO proliferative defect. Viceversa, mutants lacking the cytoplasmic domain, including the G674* truncation, act dominantly negative. Interactomic and RNA sequencing studies point to a previously unknown function of ROBO1 in RNA processing, setting the bases for future studies. Conclusions: We show for the first time that ROBO1 is necessary for MM growth and homing to the BM. Cleaved ROBO1 cytosolic domain translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO proliferative defect, possibly by participating in RNA processing. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Citation Format: Giada Bianchi, Peter G. Czarnecki, Matthew Ho, Aldo M. Roccaro, Antonio Sacco, Yawara Kawano, Annamaria Gulla, Anil Aktas Samur, Tianzeng Cheng, Kenneth Wen, Yu-Tzu Tai, Maria Moscvin, Xinchen Wu, Gulden Camci-Unal, Matteo Claudio Da VIa, Niccolo Bolli, Ruben D. Carrasco, Irene M. Ghobrial, Kenneth C. Anderson. ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB217.

Abstract Introduction: We previously showed that an imbalance between polyubiquitinated proteins awaiting proteasomal degradation and proteasome activity is a main determinant of proteasome inhibitor (PI) sensitivity in MM. While PIs have radically improved survival of MM patients, resistance is inevitable over time and understanding the molecular mechanisms underlying it is critically important to develop novel therapies to improve patient outcomes. The proteasome stress response (PSR) is an evolutionarily conserved homeostatic mechanism triggered by insufficient proteasome activity and aimed at restoring homeostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency is cleaved by the aspartic protease DDI2, translocates to the nucleus, and induces proteasome subunit gene transcription. It is unknown whether blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM. Materials and methods: We used human MM cell lines with distinct baseline sensitivity to PIs. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2 and NRF1. Cell viability or tumor growth of DDI2/NRF1 KO cells versus cells edited with a non-targeting gRNA were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate upon incubation with protein lysates from DDI2 WT, KO, or addback cells. Results: We detected full-length and cleaved NRF1 in human MM cell lines, but not other hematologic cancer cell lines, suggesting constitutively active PSR in MM. Query of the dependency map (depmap.org) shows that MM is the cell line type most highly dependent on DDI2. Consistently, DDI2 or NRF1 KO is cytotoxic alone or in combination with PI across a panel of MM cells with distinct PI sensitivity, including de novo, PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NOD SCID mice and results in prolongation of animal survival. While biallelic DDI2 or NRF1 KO monoclones could not be generated across 7 distinct MM cell lines, consistent with these genes being essential, we successfully established DDI2 KO monoclones in AMO1-VR, an isogenic AMO1 cell line variant adapted to grow in continuous bortezomib. Even in this cell line model of acquired PI resistance, DDI2 KO sensitized cells to PI carfilzomib. In this model, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome recovery after irreversible PI treatment and sensitizing to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. Conclusions: Our data show that MM cells are intrinsically dependent on an intact DDI2/NRF1 axis to survive in baseline conditions and upon proteasomal inhibition. We propose that DDI2 is an unexplored, promising molecular target for MM treatment by disrupting the PSR and exacerbating proteotoxicity. This study provides the preclinical rationale for the development of novel therapeutics targeting the DDI2/NRF1 pathway to overcome acquired and de novo PI resistance and improve patient outcomes in MM. Citation Format: Tianzeng Chen, Matthew Ho, Jenna Briere, Maria Moscvin, Peter G. Czarnecki, Kenneth C. Anderson, T. Keith Blackwell, Giada Bianchi. Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB110.

Abstract The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies. Significance: ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.

TPS8057 Background: Multiple myeloma (MM) is the second most prevalent blood cancer, representing approximately 1% of all cancers. Although overall survival has improved in recent years due to new approved agents, the vast majority of MM patients (pts) ultimately stop responding to treatment. Moreover, about a quarter of MM pts characterized as “high risk” experience limited benefit from existing treatments. Seminal genomic sequencing research efforts, such as the MMRF CoMMpass study, have highlighted that a large number of MM cases harbor potentially actionable oncogenic molecular alterations and published reports on small numbers of cases suggest that Precision Medicine (PM) interventions clinically targeting such actionable drivers may benefit MM pts. These results suggest that PM approaches in MM are possible and should be further studied clinically. To that end, we have launched MyDRUG, a master protocol aimed at developing new myeloma regimens based on individual pt’s genomics. Methods: This study is a Phase I-II, multicenter master protocol in functional high risk MM patients (i.e patients having progressed from baseline treatment within 18 months without maintenance or 3 years on maintenance) with 1-3 prior lines of therapy. Patients are screened for actionable genomic alterations on the CLIA-grade MI-ONCOSEQ platform. Patients with actionable alterations are assigned to the appropriate targeted agent used in combination with a backbone regimen ixazomib, pomalidomide and dexamethasone (IPd), whereas patients without such alterations go on an immune arm (see Table). Inclusion criteria include measurable disease (as measured by M-protein and FLC) and acceptable hematologic and metabolic functions. A maximum of 12 evaluable pts will be accrued onto the phase I portion of each arm and an additional 21 evaluable phase II pts will be accrued onto each arm for a total of 27 evaluable pts at the MTD combination dose. The primary objective of the study is to evaluate ORR per IMWG consensus criteria. Secondary objectives are to assess adverse events (AEs), progression-free survival, and overall survival (OS). Experimental correlative aims include assessing molecular or clonal response, molecular and immune signatures of resistance/response, MRD. Clinical trial information: NCT03732703. [Table: see text]

Abstract Abstract 3014 Background: Proteasome inhibitors have proven particularly effective in treatment of multiple myeloma, the second most frequent hematologic malignancy in the western world. Bortezomib, the first in class proteasome inhibitor in clinical use, was first approved in 2003 via fast FDA track, given the remarkable activity shown during phase II clinical trials. Nevertheless, more than 50% of multiple myeloma patients did not respond to single agent bortezomib when administered as second line agent. Moreover, bortezomib is only available for intravenous administration, representing a cumbersome therapy for patients, and its use is limited by significant toxicities (especially peripheral neuropathy). MLN9708 (Millennium Pharmaceuticals, Inc.), an investigational orally available, small molecule, is a potent, specific and reversible inhibitor of the 20S proteasome. It is currently under clinical investigation for the treatment of hematologic and non-hematologic malignancies. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form, and MLN2238 was used for all of the preclinical studies reported here. In vitro biochemistry studies have shown that MLN2238 has a faster dissociation rate from the proteasome compared to bortezomib, and in vivo studies of MLN2238 have shown antitumor activity in a broader range of tumor xenografts when compared to bortezomib. Given these encouraging preclinical results, we set to investigate the anti-myeloma activity of MLN2238 in vitro. Results: MLN2238 proved to have anti-proliferative and pro-apoptotic activity against a broad range of MM cell lines with EC50 at 24 hours ranging between 10 and 50 nM, even in relatively resistant MM cell lines (OPM2, DOX6, RPMI, etc.). In MM.1S cells, induction of apoptosis was time and dose dependent and related to activation of both caspase 8 and 9. When compared to MM.1S treated for 24 hours with EC50 dose of bortezomib, treatment with EC50 dose of MLN2238 resulted in the same extent of caspases cleavage occurring at an earlier time point (8-12 hours), possibly suggesting more rapid onset and/or irreversibility of apoptosis in cells treated with MLN2238. Treatment with MLN2238 was associated with early, but persistent induction of endoplasmic reticulum (ER) stress with BiP being induced 2–4 hours after treatment with EC50 dose and gradually increasing over time. While bortezomib has been associated with early induction and late decrease in proteins involved in ER stress, MLN2238 appears to induce a persistent rise in these factors, suggesting either more sustained proteasome blockade with stabilization of proteasome substrates or de-novo induction of unfolded protein response (UPR) genes. MLN2238 also proved effective in reducing phosphorylation of ERK1-2 with no overall alteration in the total ERK level, thus accounting for the observed reduction in proliferation upon treatment. Preliminary data indicate potential for additive and synergistic combination with widely used drugs, including doxorubicin and dexamethasone. Conclusion: While further clinical data are needed to establish the effectiveness of MLN2238 in the treatment of multiple myeloma, these preliminary nonclinical data, together with the favorable biochemical and pharmacokinetic properties, including oral bioavailability, make the investigational agent MLN9708 an appealing candidate for treatment of multiple myeloma. Further in vitro data could help establish whether a difference in the apoptotic mechanisms exist between MLN2238 and other proteasome inhibitors, primarily bortezomib, and could also help inform combination treatment approaches aimed at increasing effectiveness, overcoming bortezomib resistance and decreasing toxicity. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Abstract Background: Therapy with proteasome inhibitors (PIs) is an effective strategy against multiple myeloma (MM), but a significant proportion of patients is primarily resistant or eventually becomes refractory. Paradoxical activation of phospho-Akt, induction of HSP27 and excessive proteasome activity in relation to polyubiquitinated protein burden are all mechanisms for bortezomib resistance in MM cells in vitro. Preclinical data showed investigational drug MLN9708, a specific, reversible, orally bioavailable, small molecule PI, to be active in MM cell lines, patient cells and mouse models and is currently under clinical investigation. We compared the signaling pathways induced by MLN2238, the biologically active form of MLN9708, and bortezomib to outline differences that could be employed to design rational combinations to overcome resistance. Results: We performed time course experiments in MM.1S cells with EC50 doses of MLN2238 and bortezomib and analyzed signaling pathways via western blotting. Compared to bortezomib, MLN2238 induced earlier cleavage of caspase 8, 9, 3 and PARP and a significant downregulation of XIAP starting at 12 hours. Early and persistent phosphorylation of Bad, known to protect from mitochondrial apoptosis, was observed in bortezomib-, but not MLN2238-treated cells, in which phospho-Bad was reduced at 24 hours. Similarly, MLN2238 decreased phosphorylated Akt and mTOR at 24 hours, while bortezomib-treated cells only showed subtle changes. Interestingly, both drugs caused downregulation of the mTOR downstream targets phospho-p70 S6 kinase and phospho-4E-BP1, albeit of different intensity. Induction of heat shock proteins was comparable with both PI, opening the possibilities for combination therapy of MLN2238 with HSP90 inhibitors. Importantly, the inhibitory effect on phosphorylation of Akt, mTOR and Bad persisted when MM.1S cells were treated with MLN2238 in the presence of bone marrow stromal cells (BMSC). Conclusion: We observed differences in several crucial signaling pathways upon treatment of MM.1S cells with bortezomib or MLN2238. In particular, treatment with the latter inhibited phosphorylation of Bad, Akt and mTOR, suggesting a broader pro-apoptotic and anti-proliferative profile, compared to bortezomib. MLN2238 retained the capability of targeting these substrates also in the presence of BMSC. Confirmatory data on patient-derived cells are under current investigation in our lab. We believe that these data help understanding the differences in PI pharmacodynamics and bortezomib escape. This will allow us to design new drug combinations in the future that overcome clinical resistance to PIs and improve MM patient survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5669. doi:1538-7445.AM2012-5669

AbstractProtein synthesis determines sensitivity of differentiating and malignant plasma cells to proteasome inhibitors by saturating cellular proteolytic capacity.A previously unsuspected, considerable proportion of newly synthesized polypeptides are hydrolyzed rapidly by proteasomes, possibly competing with endogenous substrates and altering proteostasis. In view of the anti-cancer effects of PIs, we set out to achieve a quantitative assessment of proteasome workload in cells hallmarked by different PI sensitivity, namely, a panel of MM cells, and in a dynamic model of plasma cell differentiation, a process that confers exquisite PI sensitivity. Our results suggest that protein synthesis is a key determinant of proteasomal proteolytic burden and PI sensitivity. In different MM cells and in differentiating plasma cells, the average proteolytic work accomplished per proteasome ranges over different orders of magnitude, an unexpected degree of variability, with increased workload invariably associated to increased PI sensitivity. The unfavorable load-versus-capacity balance found in highly PI-sensitive MM lines is accounted for by a decreased total number of immunoproteasomes/cell coupled to enhanced generation of RDPs. Moreover, indicative of cause-effect relationships, attenuating general protein synthesis by the otherwise toxic agent CHX reduces PI sensitivity in activated B and in MM cells. Our data support the view that in plasma cells protein synthesis contributes to determine PI sensitivity by saturating the proteasomal degradative capacity. Quantitating protein synthesis and proteasome workload may thus prove crucial to design novel negative proteostasis regulators against cancer.

Abstract Monoclonal gammopathy of undetermined significance (MGUS) is associated with a long-term risk of progression to multiple myeloma (MM) or related malignancy. To prevent serious myeloma-related complications, lifelong annual follow-up has been recommended, but its value is unknown. We reviewed all patients from southeastern Minnesota seen at Mayo Clinic between 1973 and 2004 with MGUS who subsequently progressed to MM. Of 116 patients, 69% had optimal follow-up of MGUS. Among these, abnormalities on serial follow-up laboratory testing led to the diagnosis of MM in 16%, whereas MM was diagnosed only after serious MM-related complications in 45%. In the remaining, workup of less serious symptoms (25%), incidental finding during workup of unrelated medical conditions (11%), and unknown (3%) were the mechanisms leading to MM diagnosis. High-risk MGUS patients (≥ 1.5 g/dL and/or non-IgG MGUS) were more likely to be optimally followed (81% vs 64%), and be diagnosed with MM secondary to serial follow-up testing (21% vs 7%). This retrospective study suggests that routine annual follow-up of MGUS may not be required in low-risk MGUS. Future studies are needed to replicate and expand our findings and to determine the optimal frequency of monitoring in higher-risk MGUS patients.