Characterizing dry mass and volume changes in human multiple myeloma cells upon treatment with proteotoxic and genotoxic drugs.

Multiple myeloma (MM) is a cancer of terminally differentiated plasma cells. MM remains incurable, but overall survival of patients has progressively increased over the past two decades largely due to novel agents such as proteasome inhibitors (PI) and the immunomodulatory agents. While these therapies are highly effective, MM patients can be de novo resistant and acquired resistance with prolonged treatment is inevitable. There is growing interest in early, accurate identification of responsive versus non-responsive patients; however, limited sample availability and need for rapid assays are limiting factors. Here, we test dry mass and volume as label-free biomarkers to monitor early response of MM cells to treatment with bortezomib, doxorubicin, and ultraviolet light. For the dry mass measurement, we use two types of phase-sensitive optical microscopy techniques: digital holographic tomography and computationally enhanced quantitative phase microscopy. We show that human MM cell lines (RPMI8226, MM.1S, KMS20, and AMO1) increase dry mass upon bortezomib treatment. This dry mass increase after bortezomib treatment occurs as early as 1 h for sensitive cells and 4 h for all tested cells. We further confirm this observation using primary multiple myeloma cells derived from patients and show that a correlation exists between increase in dry mass and sensitivity to bortezomib, supporting the use of dry mass as a biomarker. The volume measurement using Coulter counter shows a more complex behavior; RPMI8226 cells increase the volume at an early stage of apoptosis, but MM.1S cells show the volume decrease typically observed with apoptotic cells. Altogether, this cell study presents complex kinetics of dry mass and volume at an early stage of apoptosis, which may serve as a basis for the detection and treatment of MM cells.

Mutations in the alternative complement pathway in multiple myeloma patients with carfilzomib-induced thrombotic microangiopathy.

Thrombotic microangiopathy (TMA) has been reported to occur in multiple myeloma (MM) patients in association with treatment with carfilzomib, an irreversible proteasome inhibitor (PI). The hallmark of TMA is vascular endothelial damage leading to microangiopathic hemolytic anemia, platelet consumption, fibrin deposition and small-vessel thrombosis with resultant tissue ischemia. The molecular mechanisms underlying carfilzomib-associated TMA are not known. Germline mutations in the complement alternative pathway have been recently shown to portend increased risk for the development of atypical hemolytic uremic syndrome (aHUS) and TMA in the setting of allogeneic stem cell transplant in pediatric patients. We hypothesized that germline mutations in the complement alternative pathway may similarly predispose MM patients to carfilzomib-associated TMA. We identified 10 MM patients with a clinical diagnosis of TMA in the context of carfilzomib treatment and assessed for the presence of germline mutations in the complement alternative pathway. Ten, matched MM patients exposed to carfilzomib but without clinical TMA were used as negative controls. We identified a frequency of deletions in the complement Factor H genes 3 and 1 (delCFHR3-CFHR1) and genes 1 and 4 (delCFHR1-CFHR4) in MM patients with carfilzomib-associated TMA that was higher as compared to the general population and matched controls. Our data suggest that complement alternative pathway dysregulation may confer susceptibility to vascular endothelial injury in MM patients and predispose to development of carfilzomib-associated TMA. Larger, retrospective studies are needed to evaluate whether screening for complement mutations may be indicated to properly counsel patients about TMA risk with carfilzomib use.

Targeting LAG3/GAL-3 to overcome immunosuppression and enhance anti-tumor immune responses in multiple myeloma.

Immune profiling in patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM) provides the framework for developing novel immunotherapeutic strategies. Here, we demonstrate decreased CD4+ Th cells, increased Treg and G-type MDSC, and upregulation of immune checkpoints on effector/regulatory and CD138+ cells in MM patients, compared MGUS/SMM patients or healthy individuals. Among the checkpoints profiled, LAG3 was most highly expressed on proliferating CD4+ Th and CD8+ Tc cells in MM patients BMMC and PBMC. Treatment with antibody targeting LAG3 significantly enhanced T cells proliferation and activities against MM. XBP1/CD138/CS1-specific CTL generated in vitro displayed anti-MM activity, which was further enhanced following anti-LAG3 treatment, within the antigen-specific memory T cells. Treg and G-type MDSC weakly express LAG3 and were minimally impacted by anti-LAG3. CD138+ MM cells express GAL-3, a ligand for LAG3, and anti-GAL-3 treatment increased MM-specific responses, as observed for anti-LAG3. Finally, we demonstrate checkpoint inhibitor treatment evokes non-targeted checkpoints as a cause of resistance and propose combination therapeutic strategies to overcome this resistance. These studies identify and validate blockade of LAG3/GAL-3, alone or in combination with immune strategies including XBP1/CD138/CS1 multipeptide vaccination, to enhance anti-tumor responses and improve patient outcome in MM.