In collaboration with Dr. Landgren's group, we have investigated proteasome changes in neoplastic plasma cells from bone marrows of multiple myeloma (MM) patients. Some of the hallmarks of cancer, such as rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of MM to 20S proteasome inhibitors, such as carfilzomib. However, MM patients invariably acquire resistance to these drugs. The mechanism is not well understood. We have devised a novel flow cytometric assay to monitor proteasome subunit (19S and 20S) levels pre and post carfilzomib therapy. Surprisingly, we have discovered paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits. In MM patients, lower 19S levels predicted a diminished response to carfilzomib-based therapies while 20S levels had no predictive effect. To further understand this finding, we collaborated with Dr. Kampmann group from UCSF, who utilized a next-generation shRNA platform to show that different proteostasis factors, including chaperones and stress-response regulators, controll the response to carfilzomib. In in vitro experiments, 19S proteasome regulator knockdown induced resistance to carfilzomib in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. Together, our findings suggest that an understanding of network rewiring can inform development of new combination therapies to overcome drug resistance. In a separate study, we have described a previously unrecognized disease entity that we have named Monoclonal gammopathy-associated pure red cell aplasia. Pure red cell aplasia (PRCA) is a rare disorder characterized by absence of erythroid precursors in the bone marrow and severe normochromic, normocytic anaemia with reticulocytopenia. The cause of red cell aplasia is unknown in a subset of patients who are labeled idiopathic PRCA. We studied bone marrow changes in idiopathic PRCA patients and discovered that among 51 patients, 12 (24%) patients had increased clonal plasma cells and fulfilled criteria for either monoclonal gammopathy, monoclonal gammopathy of undetermined significance or smouldering multiple myeloma, with presence of monoclonal protein or abnormal serum free light chains. The marrow biopsies showed atypical bone marrow features including hypercellularity, fibrosis and clonal plasmacytosis. Three of these patients have been treated so far with anti-myeloma based therapeutics and responded with reticulocyte recovery and clinical transfusion independence, suggesting plasma cells play a key role in the pathogenesis of this specific monoclonal gammopathy-associated PRCA. The incidence and importance of bone marrow involvement and/or early bone lesions in multiple myeloma (MM) precursor diseases is largely unknown. We have conducted a study to prospectively compare the sensitivity of several imaging modalities in monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and MM and compare to bone marrow morphological findings. Thirty patients (10 each with MGUS, SMM and MM) were evaluated with skeletal survey, 18FFDG-PET/CT, 18FNaF-PET/CT and morphologic dynamic contrast enhanced (DCE)-MRI. An additional 16 SMM patients had skeletal surveys and FDG-PET/CT. Among MGUS patients diagnosed based on the WHO criteria, DCE-MRI found only one focal marrow abnormality; other evaluations were negative. Among 26 SMM patients, five (19%) were re-classified as MM based on lytic bone lesions on CT and six had unifocal or diffuse marrow abnormality. Among MM, marrow abnormalities were observed on FDG-PET/CT in 8/10 patients and on DCE-MRI in nine evaluable patients. Abnormal NaF uptake was observed only in MM patients with lytic lesions on CT, providing no additional clinical information. In collaboration with Dr. Wiestner's group, we have investigated chronic lymphocytic leukemia tumor-microenvironment interactions in patients treated with Ibrutinib, a Bruton tyrosine kinase inhibitor. Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the effects of ibrutinib on the tumor microenvironment. Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed. Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4(+)T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4(+)T cells in vitro. Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells. We conclude that in conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL.
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