Expression of the chemokine receptor CCR1 promotes the dissemination of multiple myeloma plasma cells in vivo

Multiple myeloma (MM) disease progression is dependent on the ability of MM plasma cells (PCs) to egress from the bone marrow (BM), enter the circulation and disseminate to distal BM sites. Expression of the chemokine CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome CXCL12-mediated retention to enable dissemination are poorly understood. We have previously identified that treatment with the CCR1 ligand CCL3 inhibits the response to CXCL12 in MM cell lines, suggesting that CCL3/CCR1 signalling may enable egress of MM PC from the BM. Here, we demonstrated that CCR1 expression was an independent prognostic indicator in newly diagnosed MM patients. Furthermore, we showed that CCR1 is a crucial driver of dissemination in vivo, with CCR1 expression in the murine MM cell line 5TGM1 being associated with an increased incidence of bone and splenic disseminated tumours in C57BL/KaLwRij mice. Furthermore, we demonstrated that CCR1 knockout in the human myeloma cell line OPM2 resulted in a >95% reduction in circulating MM PC numbers and BM and splenic tumour dissemination following intratibial injection in NSG mice. Therapeutic targeting of CCR1 with the inhibitor CCX9588 significantly reduced OPM2 or RPMI-8226 dissemination in intratibial xenograft models. Collectively, our findings suggest a novel role for CCR1 as a critical driver of BM egress of MM PCs during tumour dissemination. Furthermore, these data suggest that CCR1 may represent a potential therapeutic target for the prevention of MM tumour dissemination.

Expression of the chemokine receptor CCR1 decreases sensitivity to bortezomib in multiple myeloma cell lines.

BACKGROUND: The proteasome inhibitor bortezomib is one of the primary therapies used for the haematological malignancy multiple myeloma (MM). However, intrinsic or acquired resistance to bortezomib, via mechanisms that are not fully elucidated, is a barrier to successful treatment in many patients. Our previous studies have shown that elevated expression of the chemokine receptor CCR1 in MM plasma cells in newly diagnosed MM patients is associated with poor prognosis. Here, we hypothesised that the poor prognosis conferred by CCR1 expression is, in part, due to a CCR1-mediated decrease in MM plasma cell sensitivity to bortezomib. METHODS: In order to investigate the role of CCR1 in MM cells, CCR1 was knocked out in human myeloma cell lines OPM2 and U266 using CRISPR-Cas9. Additionally, CCR1 was overexpressed in the mouse MM cell line 5TGM1. The effect of bortezomib on CCR1 knockout or CCR1-overexpressing cells was then assessed by WST-1 assay, with or without CCL3 siRNA knockdown or addition of recombinant human CCL3. NSG mice were inoculated intratibially with OPM2-CCR1KO cells and were treated with 0.7 mg/kg bortezomib or vehicle twice per week for 3 weeks and GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. The effect of CCR1 overexpression or knockout on unfolded protein response pathways was assessed using qPCR for ATF4, HSPA5, XBP1, ERN1 and CHOP and Western blot for IRE1α and p-Jnk. RESULTS: Using CCR1 overexpression or CRIPSR-Cas9-mediated CCR1 knockout in MM cell lines, we found that CCR1 expression significantly decreases sensitivity to bortezomib in vitro, independent of the CCR1 ligand CCL3. In addition, CCR1 knockout rendered the human MM cell line OPM2 more sensitive to bortezomib in an intratibial MM model in NSG mice in vivo. Moreover, CCR1 expression negatively regulated the expression of the unfolded protein response receptor IRE1 and downstream target gene XBP1, suggesting this pathway may be responsible for the decreased bortezomib sensitivity of CCR1-expressing cells. CONCLUSIONS: Taken together, these studies suggest that CCR1 expression may be associated with decreased response to bortezomib in MM cell lines.

Next batter up! Targeting cancers with KRAS-G12D mutations.

KRAS is the most frequently mutated oncogene in cancer. Activating mutations in codon 12, especially G12D, have the highest prevalence across a range of carcinomas and adenocarcinomas. With inhibitors to KRAS-G12D now entering clinical trials, understanding the biology of KRAS-G12D cancers, and identifying biomarkers that predict therapeutic response is crucial. In this Review, we discuss the genomics and biology of KRAS-G12D adenocarcinomas, including histological features, transcriptional landscape, the immune microenvironment, and how these factors influence response to therapy. Moreover, we explore potential therapeutic strategies using novel G12D inhibitors, leveraging knowledge gained from clinical trials using G12C inhibitors.

PTPN2 elicits cell autonomous and non-cell autonomous effects on antitumor immunity in triple-negative breast cancer.

The tumor-suppressor PTPN2 is diminished in a subset of triple-negative breast cancers (TNBCs). Paradoxically, PTPN2-deficiency in tumors or T cells in mice can facilitate T cell recruitment and/or activation to promote antitumor immunity. Here, we explored the therapeutic potential of targeting PTPN2 in tumor cells and T cells. PTPN2-deficiency in TNBC associated with T cell infiltrates and PD-L1 expression, whereas low PTPN2 associated with improved survival. PTPN2 deletion in murine mammary epithelial cells TNBC models, did not promote tumorigenicity but increased STAT-1-dependent T cell recruitment and PD-L1 expression to repress tumor growth and enhance the efficacy of anti-PD-1. Furthermore, the combined deletion of PTPN2 in tumors and T cells facilitated T cell recruitment and activation and further repressed tumor growth or ablated tumors already predominated by exhausted T cells. Thus, PTPN2-targeting in tumors and/or T cells facilitates T cell recruitment and/or alleviates inhibitory constraints on T cells to combat TNBC.

PTP1B Is an Intracellular Checkpoint that Limits T-cell and CAR T-cell Antitumor Immunity.

Immunotherapies aimed at alleviating the inhibitory constraints on T cells have revolutionized cancer management. To date, these have focused on the blockade of cell-surface checkpoints such as PD-1. Herein we identify protein tyrosine phosphatase 1B (PTP1B) as an intracellular checkpoint that is upregulated in T cells in tumors. We show that increased PTP1B limits T-cell expansion and cytotoxicity to contribute to tumor growth. T cell-specific PTP1B deletion increased STAT5 signaling, and this enhanced the antigen-induced expansion and cytotoxicity of CD8+ T cells to suppress tumor growth. The pharmacologic inhibition of PTP1B recapitulated the T cell-mediated repression of tumor growth and enhanced the response to PD-1 blockade. Furthermore, the deletion or inhibition of PTP1B enhanced the efficacy of adoptively transferred chimeric antigen receptor (CAR) T cells against solid tumors. Our findings identify PTP1B as an intracellular checkpoint whose inhibition can alleviate the inhibitory constraints on T cells and CAR T cells to combat cancer. SIGNIFICANCE: Tumors subvert antitumor immunity by engaging checkpoints that promote T-cell exhaustion. Here we identify PTP1B as an intracellular checkpoint and therapeutic target. We show that PTP1B is upregulated in intratumoral T cells and that its deletion or inhibition enhances T-cell antitumor activity and increases CAR T-cell effectiveness against solid tumors. This article is highlighted in the In This Issue feature, p. 587.

Tumour Dissemination in Multiple Myeloma Disease Progression and Relapse: A Potential Therapeutic Target in High-Risk Myeloma.

Multiple myeloma (MM) is a plasma cell (PC) malignancy characterised by the presence of MM PCs at multiple sites throughout the bone marrow. Increased numbers of peripheral blood MM PCs are associated with rapid disease progression, shorter time to relapse and are a feature of advanced disease. In this review, the current understanding of the process of MM PC dissemination and the extrinsic and intrinsic factors potentially driving it are addressed through analysis of patient-derived MM PCs and MM cell lines as well as mouse models of homing and dissemination. In addition, we discuss how patient cytogenetic subgroups that present with highly disseminated disease, such as t(4;14), t(14;16) and t(14;20), suggest that intrinsic properties of MM PC influence their ability to disseminate. Finally, we discuss the possibility of using therapeutic targeting of tumour dissemination to slow disease progression and prevent overt relapse.

HIF-2α Promotes Dissemination of Plasma Cells in Multiple Myeloma by Regulating CXCL12/CXCR4 and CCR1.

Disease progression and relapse in multiple myeloma is dependent on the ability of the multiple myeloma plasma cells (PC) to reenter the circulation and disseminate throughout the bone marrow. Increased bone marrow hypoxia is associated with increased recirculation of multiple myeloma PCs. Accordingly, we hypothesized that during chronic hypoxia, activation of HIF-2α may overcome the bone marrow retention signal provided by stromal-derived CXCL12, thereby enabling dissemination of multiple myeloma PCs. Here we demonstrate that HIF-2α upregulates multiple myeloma PC CXCL12 expression, decreasing migration toward CXCL12 and reducing adhesion to mesenchymal stromal cells in vitro We also found that HIF-2α strongly induced expression of the chemokine receptor CCR1 in multiple myeloma PCs. CCR1 activation potently induces multiple myeloma PC migration toward CCL3 while abrogating the multiple myeloma PC migratory response to CXCL12. In addition, increased CCR1 expression by multiple myeloma PCs conferred poor prognosis in newly diagnosed multiple myeloma patients and was associated with an increase in circulating multiple myeloma PCs in these patients. Taken together, our results suggest a role for hypoxia-mediated CCR1 upregulation in driving the egress of multiple myeloma PCs from the bone marrow. Targeting CCR1 may represent a novel strategy to prevent dissemination and overt relapse in multiple myeloma. Cancer Res; 77(20); 5452-63. ©2017 AACR.