Rakesh Bam

Role of Bruton's tyrosine kinase in myeloma cell migration and induction of bone disease

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Brutons tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B‐lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL)−6– or stroma–dependent cell lines and osteoclasts. SDF‐1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM‐A13, reduced their migration toward stromal cell‐derived factor‐1 (SDF‐1). Pretreatment with LFM‐A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID‐rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF‐1 but had no effect on short‐term growth. BTK expression was correlated with cell‐surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell‐surface CXCR4‐expressing myeloma cells. BTK was not upregulated by IL‐6 while its inhibition had no effect on IL‐6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell‐surface CXCR4 and migrated toward SDF‐1. LFM‐A13 suppressed migration and differentiation of osteoclast precursors as well as bone‐resorbing activity of mature osteoclasts. In primary myeloma‐bearing SCID‐rab mice, LFM‐A13 inhibited osteoclast activity, prevented myeloma‐induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell‐surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma‐induced bone disease. Am. J. Hematol. 88:463–471, 2013.

CYR61/CCN1 overexpression in the myeloma microenvironment is associated with superior survival and reduced bone disease

Secreted protein CCN1, encoded by CYR61, is involved in wound healing, angiogenesis, and osteoblast differentiation. We identified CCN1 as a microenvironmental factor produced by mesenchymal cells and overexpressed in bones of a subset of patients with monoclonal gammopathy of undetermined significance (MGUS), asymptomatic myeloma (AMM), and multiple myeloma (MM). Our analysis showed that overexpression of CYR61 was independently associated with superior overall survival of MM patients enrolled in our Total Therapy 3 protocol. Moreover, elevated CCN1 was associated with a longer time for MGUS/AMM to progress to overt MM. During remission from MM, high levels of CCN1 were associated with superior progression-free and overall survival and stratified patients with molecularly defined high-risk MM. Recombinant CCN1 directly inhibited in vitro growth of MM cells, and overexpression of CYR61 in MM cells reduced tumor growth and prevented bone destruction in vivo in severe combined immunodeficiency-hu mice. Signaling through αvβ3 was required for CCN1 prevention of bone disease. CYR61 expression may signify early perturbation of the microenvironment before conversion to overt MM and may be a compensatory mechanism to control MM progression. Therapeutics that upregulate CYR61 should be investigated for treating MM bone disease.

Identification of Doxorubicin as an Inhibitor of the IRE1α-XBP1 Axis of the Unfolded Protein Response

Activation of the IRE1α-XBP1 branch of the unfolded protein response (UPR) has been implicated in multiple types of human cancers, including multiple myeloma (MM). Through an in silico drug discovery approach based on protein-compound virtual docking, we identified the anthracycline antibiotic doxorubicin as an in vitro and in vivo inhibitor of XBP1 activation, a previously unknown activity for this widely utilized cancer chemotherapeutic drug. Through a series of mechanistic and phenotypic studies, we showed that this novel activity of doxorubicin was not due to inhibition of topoisomerase II (Topo II). Consistent with its inhibitory activity on the IRE1α-XBP1 branch of the UPR, doxorubicin displayed more potent cytotoxicity against MM cell lines than other cancer cell lines that have lower basal IRE1α-XBP1 activity. In addition, doxorubicin significantly inhibited XBP1 activation in CD138+ tumor cells isolated from MM patients. Our findings suggest that the UPR-modulating activity of doxorubicin may be utilized clinically to target IRE1α–XBP1-dependent tumors such as MM.

Role of Bruton's tyrosine kinase (BTK) in growth and metastasis of INA6 myeloma cells

Brutons tyrosine kinase (BTK) and the chemokine receptor CXCR4 are linked in various hematologic malignancies. The aim of the study was to understand the role of BTK in myeloma cell growth and metastasis using the stably BTK knockdown luciferase-expressing INA6 myeloma line. BTK knockdown had reduced adhesion to stroma and migration of myeloma cells toward stromal cell-derived factor-1. BTK knockdown had no effect on short-term in vitro growth of myeloma cells, although clonogenicity was inhibited and myeloma cell growth was promoted in coculture with osteoclasts. In severe combined immunodeficient-rab mice with contralaterally implanted pieces of bones, BTK knockdown in myeloma cells promoted their proliferation and growth in the primary bone but suppressed metastasis to the contralateral bone. BTK knockdown myeloma cells had altered the expression of genes associated with adhesion and proliferation and increased mammalian target of rapamycin signaling. In 176 paired clinical samples, BTK and CXCR4 expression was lower in myeloma cells purified from a focal lesion than from a random site. BTK expression in random-site samples was correlated with proportions of myeloma cells expressing cell surface CXCR4. Our findings highlight intratumoral heterogeneity of myeloma cells in the bone marrow microenvironment and suggest that BTK is involved in determining proliferative, quiescent or metastatic phenotypes of myeloma cells.

Abstract 1648: Primary myeloma plasma cells are capable of growth in adult, normal whole human bone marrow environment .

Multiple myeloma (MM) is a neoplasm of low proliferative plasma cells that are highly dependent on bone marrow (BM) microenvironment for survival and growth. Primary human MM cells do not survive in conditions designed for cell lines and cell lines do not represent the primary cell heterogeneity and are microenvironment independent. Current in vitro and in vivo systems for primary human MM are limited to co-culture with specific BM cell type or growth in immunodeficient animal model. We and others have demonstrated the ability of the recognizable mature, C138+ or CD45-/CD38+ MM cells to produce MM in those experimental systems. The aim of the study was to establish and test the survival and growth of primary MM plasma cells in long term culture with a complete adult normal BM. This system is different from the autologous BM culture that is already affected by the disease. Whole BM cells from healthy donors were cultured in αMEM medium supplemented with 10% FBS and 10% serum from MM patients. Following 7-9 days the cultures were composed of adherent and nonadherent cellular compartments. The nonadherent compartment contained typical BM hematopoietic cells such monocytes, B and T lymphocytes and NK cells as assessed by flow cytometry, while the adherent compartment contained cells that morphologically resemble macrophages, osteoclasts, megakaryocytes and fibroblast-like cells. At this culture stage, CD138-selected MM cells from 13 patients were added to the BM cultures and survival and growth of MM cells were determined after 7 days by assessing proportion of CD45 low/intermediate /CD38 high MM plasma cells among total number of nonadherent cells. Low proportions of normal plasma cells (0.1-1.7%) were also detected in the cultures throughout the duration of experiments. In additional study, we used luciferase-expressing IL6-dependent INA6 MM cells and stroma-dependent MM line to assess MM growth within the whole BM cultures using bioluminescence analysis. Cell viability was constantly high (>90%) in the primary MM cell-normal BM cultures. Subsets of primary MM plasma cells were survived and detected in all culture cases while in 9 of 13 experiments, number of primary MM plasma cells was heterogeneously increased by 63±19% (p Citation Format: Rakesh Bam, Sathisha Upparahalli Venkateshaiah, Xin Li, Sharmin Khan, Wen Ling, Bart Barlogie, Joshua Epstein, Shmuel Yaccoby. Primary myeloma plasma cells are capable of growth in adult, normal whole human bone marrow environment . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1648. doi:10.1158/1538-7445.AM2013-1648

Inhibition of IRE1 results in decreased scar formation

Wound healing is characterized by the production of large amounts of protein necessary to replace lost cellular mass and extracellular matrix. The unfolded protein response (UPR) is an important adaptive cellular response to increased protein synthesis. One of the main components of the UPR is IRE1, an endoplasmic reticulum transmembrane protein with endonuclease activity that produces the activated form of the transcription factor XBP1. Using luciferase reporter mice for Xbp1 splicing, we showed that IRE1 was up‐regulated during excisional wound healing at the time in wound healing consistent with that of the proliferative phase, when the majority of protein synthesis for cellular proliferation and matrix deposition occurs. Furthermore, using a small molecule inhibitor of IRE1 we demonstrated that inhibition of IRE1 led to decreased scar formation in treated mice. Results were recapitulated in a hypertrophic scar mouse model. These data help provide a cellular pathway to target in the treatment of hypertrophic scarring and keloid disorders.