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Supplementary MethodsSupplementary Methods descript detailed methods of Bioluminescence imaging, ELISA, Flow cytometry, Isolation and culture of Pre-OBs with 5TGM1-Luc MM cells, Generation and sorting of MDSCs, Cell proliferation assays, Treatment of MM cells with OB-Runx2+/+ and OB-Runx2-/- BMS in vitro, and Western blotting.

In multiple myeloma, abnormal plasma cells accumulate and proliferate in the bone marrow. Recently, we observed that Runx2, a bone-specific transcription factor, is highly expressed in multiple myeloma cells and is a major driver of multiple myeloma progression in bone. The primary goal of the present study was to identify Runx2-targeting miRNAs that can reduce tumor growth. Expression analysis of a panel of miRNAs in multiple myeloma patient specimens, compared with healthy control specimens, revealed that metastatic multiple myeloma cells express low levels of miR-342 and miR-363 but high levels of Runx2. Reconstituting multiple myeloma cells (CAG) with miR-342 and miR-363 reduced the abundance of Runx2 and the expression of metastasis-promoting Runx2 target genes RANKL and DKK1, and suppressed Runx2 downstream signaling pathways Akt/β-catenin/survivin, which are required for multiple myeloma tumor progression. Intravenous injection of multiple myeloma cells (5TGM1), stably overexpressing miR-342 and miR-363 alone or together, into syngeneic C57Bl/KaLwRij mice resulted in a significant suppression of 5TGM1 cell growth, decreased osteoclasts and increased osteoblasts, and increased antitumor immunity in the bone marrow, compared with mice injected with 5TGM1 cells expressing a miR-Scramble control. In summary, these results demonstrate that enhanced expression of miR-342 and miR-363 in multiple myeloma cells inhibits Runx2 expression and multiple myeloma growth, decreases osteolysis, and enhances antitumor immunity. Thus, restoring the function of Runx2-targeting by miR-342 and miR-363 in multiple myeloma cells may afford a therapeutic benefit by preventing multiple myeloma progression.Implications: miR-342 and miR-363–mediated downregulation of Runx2 expression in multiple myeloma cells prevents multiple myeloma progression. Mol Cancer Res; 16(7); 1138–48. ©2018 AACR.

Antibodies used for Western blotting and FACS.

Supplementary Figure S1-S6Supplementary Figure S1 shows tumor burden in the mice before BTZ treatment started (S1A), MM cell growth and apoptosis after co-cultured with BMS or pre-OBs of OB-Runx2-/- or OB-Runx2+/+ mice (S1B-F), and the proportion of PMN-MDSCs and M-MDSCs in the BM of Runx2-/- or OB-Runx2+/+ mice after 4-week treatment with BTZ (S1G-H).Supplementary Figure S2 shows tumor burden in the mice before SRI31277 and BTZ treatment (S2A) and the proportion of PMN-MDSCs and M-MDSCs in the BM of Runx2-/- or OB-Runx2+/+ mice after 4-week treatment with SRI31277 and BTZ (S2B-C).Supplementary Figure S3 shows tumor burden in the mice before 5-Fu and BTZ treatment.Supplementary Figure S4 shows the impact of GEM treatment on BTZ resistance of MM cells in OB-Runx2-/- mice.Supplementary Figure S5 shows the proportion of PMN-MDSCs and M-MDSCs in the BM of Runx2-/- or OB-Runx2+/+ mice after 4-week treatment with 5-Fu and BTZ.Supplementary Figure S6 shows the impact of GEM and BTZ treatment on MDSCs and CD8 T cells.

5TGM1 cells stably overexpressing miR-342 and miR-363 were established by lentiviral vector transduction.

Supplementary Table S1Supplementary Table S1 shows the company's name and the product numbers of Bortezomib, 5-Fluorouracil and Gemcitabine as well as the company's name and the product numbers of all antibodies listed in table S1, which we purchased and used for this study.

Multiple myeloma is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib is one of the most effective first-line chemotherapeutic drugs for multiple myeloma; however, 15% to 20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that multiple myeloma cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OB), and that this OB-Runx2 deficiency induces a cytokine-rich and immunosuppressive microenvironment in the BM. In the current study, we assessed the impact of OB-Runx2 deficiency on the outcome of bortezomib treatment using OB-Runx2+/+ and OB-Runx2−/− mouse models of multiple myeloma. In vitro and in vivo experiments revealed that OB-Runx2 deficiency induces multiple myeloma cell resistance to bortezomib via the upregulation of immunosuppressive myeloid-derived suppressor cells (MDSCs), downregulation of cytotoxic T cells, and activation of TGFβ1 in the BM. In multiple myeloma tumor-bearing OB-Runx2−/− mice, treatment with SRI31277, an antagonist of thrombospondin-1 (TSP-1)–mediated TGFβ1 activation, reversed the BM immunosuppression and significantly reduced tumor burden. Furthermore, treatment with SRI31277 combined with bortezomib alleviated multiple myeloma cell resistance to bortezomib-induced apoptosis caused by OB-Runx2 deficiency in cocultured cells and produced a synergistic effect on tumor burden in OB-Runx2−/− mice. Depletion of MDSCs by 5-fluorouracil or gemcitabine similarly reversed the immunosuppressive effects and bortezomib resistance induced by OB-Runx2 deficiency in tumor-bearing mice, indicating the importance of the immune environment for drug resistance and suggesting new strategies to overcome bortezomib resistance in the treatment of multiple myeloma.

The Supplementary Figures show the characterization of OB-Runx2 mouse model and the results of analysis of BM immunity after depletion of MDSCs by 5-FU.

The Supplementary Table S1 provides the information about all the antibodies used in flow cytometry analysis and immunostaining in this study.

Detailed and supplementary methods

List of significant gene symbols

SF1: IL-26 protein or mRNA expression in human cell lines and clinical samples; SF2: Single-cell profiling of CD4+ TH17 cells in TNBC patients; SF3: Confirmation of IL-26 knockdown in MDA-MB-231 cells; SF4: In vitro proliferation and in vivo metastasis of IL-26 knockdown MDA-MB-231 cells; SF5: Gene expression profiling of IL-26 knockdown MDA-MB-231 tumors; SF6: IL-26 knockdown in SUM159 and MDA-MB-468 cells and in vitro phenotyping; SF7: Western blot confirmation of IL-26 knock-in E0771 cells and IL-26 upregulation after growth in vivo; SF8: IL-26 knock-in E0771 tumors progress more rapidly than control in an immune competent setting; SF9: Stat3 activation by IL-26 is dependent on human IL10RB and IL20RA; SF10: DNA-bound IL-26 elicits inflammatory signaling in human and mouse immune cells; SF11: Confirmation of mouse neutrophil enrichment; SF12: Confirmation of Anti-Ly6G antibody depletion of mouse neutrophils; SF13: Inflammatory cytokine expression in MDA-MB-231 parental and MDA-MB-231 LM2 lung-metastatic subclone; SF14: CRISPR knockdown of IL-6, IL-8, and Cxcl1 in MDA-MB-231 LM2 CRISPR 3x cell lines/lung metastases and correlation of CRISPR 3x primary tumor volume with metastasis; SF15: IL-26 signature and Neutrophil enrichment score are significantly correlated in METABRIC dataset; SF16: T cell ELISPOT response to IL-26 vaccination in vivo.

Significant hits identified in CRISPR screen

The Supplementary Methods provide more detailed information on OB-Runx2 mouse model and in vivo/ in vitro experiments.

CRISPR screen results

IL26 is a unique amphipathic member of the IL10 family of cytokines that participates in inflammatory signaling through a canonical receptor pathway. It also directly binds DNA to facilitate cellular transduction and intracellular inflammatory signaling. Although IL26 has almost no described role in cancer, our in vivo screen of inflammatory and cytokine pathway genes revealed IL26 to be one of the most significant inflammatory mediators of mammary engraftment and lung metastatic growth in triple-negative breast cancer (TNBC). Examination of human breast cancers demonstrated elevated IL26 transcripts in TNBC specimens, specifically in tumor cells as well as in Th17 CD4+ T cells within clinical TNBC specimens. IL26 did not have an autocrine effect on human TNBC cells, but rather its effect on engraftment and growth in vivo required neutrophils. IL26 enhanced mouse-derived DNA induction of inflammatory cytokines, which were collectively important for mammary and metastatic lung engraftment. To neutralize this effect, we developed a novel IL26 vaccine to stimulate antibody production and suppress IL26-enhanced engraftment in vivo, suggesting that targeting this inflammatory amplifier could be a unique means to control cancer-promoting inflammation in TNBC and other autoimmune diseases. Thus, we identified IL26 as a novel key modulator of TNBC metastasis and a potential therapeutic target in TNBC as well as other diseases reliant upon IL26-mediated inflammatory stimulation.Significance:These findings identify IL26 as a unique, clinically relevant, inflammatory amplifier that enhances TNBC engraftment and dissemination in association with neutrophils, which has potential as a therapeutic target.

Multiple myeloma is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib is one of the most effective first-line chemotherapeutic drugs for multiple myeloma; however, 15% to 20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that multiple myeloma cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OB), and that this OB-Runx2 deficiency induces a cytokine-rich and immunosuppressive microenvironment in the BM. In the current study, we assessed the impact of OB-Runx2 deficiency on the outcome of bortezomib treatment using OB-Runx2+/+ and OB-Runx2−/− mouse models of multiple myeloma. In vitro and in vivo experiments revealed that OB-Runx2 deficiency induces multiple myeloma cell resistance to bortezomib via the upregulation of immunosuppressive myeloid-derived suppressor cells (MDSCs), downregulation of cytotoxic T cells, and activation of TGFβ1 in the BM. In multiple myeloma tumor-bearing OB-Runx2−/− mice, treatment with SRI31277, an antagonist of thrombospondin-1 (TSP-1)–mediated TGFβ1 activation, reversed the BM immunosuppression and significantly reduced tumor burden. Furthermore, treatment with SRI31277 combined with bortezomib alleviated multiple myeloma cell resistance to bortezomib-induced apoptosis caused by OB-Runx2 deficiency in cocultured cells and produced a synergistic effect on tumor burden in OB-Runx2−/− mice. Depletion of MDSCs by 5-fluorouracil or gemcitabine similarly reversed the immunosuppressive effects and bortezomib resistance induced by OB-Runx2 deficiency in tumor-bearing mice, indicating the importance of the immune environment for drug resistance and suggesting new strategies to overcome bortezomib resistance in the treatment of multiple myeloma.

IL26 is a unique amphipathic member of the IL10 family of cytokines that participates in inflammatory signaling through a canonical receptor pathway. It also directly binds DNA to facilitate cellular transduction and intracellular inflammatory signaling. Although IL26 has almost no described role in cancer, our in vivo screen of inflammatory and cytokine pathway genes revealed IL26 to be one of the most significant inflammatory mediators of mammary engraftment and lung metastatic growth in triple-negative breast cancer (TNBC). Examination of human breast cancers demonstrated elevated IL26 transcripts in TNBC specimens, specifically in tumor cells as well as in Th17 CD4+ T cells within clinical TNBC specimens. IL26 did not have an autocrine effect on human TNBC cells, but rather its effect on engraftment and growth in vivo required neutrophils. IL26 enhanced mouse-derived DNA induction of inflammatory cytokines, which were collectively important for mammary and metastatic lung engraftment. To neutralize this effect, we developed a novel IL26 vaccine to stimulate antibody production and suppress IL26-enhanced engraftment in vivo, suggesting that targeting this inflammatory amplifier could be a unique means to control cancer-promoting inflammation in TNBC and other autoimmune diseases. Thus, we identified IL26 as a novel key modulator of TNBC metastasis and a potential therapeutic target in TNBC as well as other diseases reliant upon IL26-mediated inflammatory stimulation. Significance: These findings identify IL26 as a unique, clinically relevant, inflammatory amplifier that enhances TNBC engraftment and dissemination in association with neutrophils, which has potential as a therapeutic target.

Multiple myeloma is a plasma cell malignancy that thrives in the bone marrow (BM), with frequent progression to new local and distant bone sites. Our previous studies demonstrated that multiple myeloma cells at primary sites secrete soluble factors and suppress osteoblastogenesis via the inhibition of Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OB) in new bone sites, prior to the arrival of metastatic tumor cells. However, it is unknown whether OB-Runx2 suppression in new bone sites feeds back to promote multiple myeloma dissemination to and progression in these areas. Hence, we developed a syngeneic mouse model of multiple myeloma in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2−/− mice) to study the effect of OB-Runx2 deficiency on multiple myeloma progression in new bone sites. In vivo studies with this model demonstrated that OB-Runx2 deficiency attracts multiple myeloma cells and promotes multiple myeloma tumor growth in bone. Mechanistic studies further revealed that OB-Runx2 deficiency induces an immunosuppressive microenvironment in BM that is marked by an increase in the concentration and activation of myeloid-derived suppressor cells (MDSC) and the suppression and exhaustion of cytotoxic CD8+ T cells. In contrast, MDSC depletion by either gemcitabine or 5-fluorouracil treatment in OB-Runx2−/− mice prevented these effects and inhibited multiple myeloma tumor growth in BM. These novel discoveries demonstrate that OB-Runx2 deficiency in new bone sites promotes multiple myeloma dissemination and progression by increasing metastatic cytokines and MDSCs in BM and inhibiting BM immunity. Importantly, MDSC depletion can block multiple myeloma progression promoted by OB-Runx2 deficiency. Significance: This study demonstrates that Runx2 deficiency in immature osteoblasts at distant bone sites attracts myeloma cells and allows myeloma progression in new bone sites via OB-secreted metastatic cytokines and MDSC-mediated suppression of bone marrow immunity.

A significant portion of breast cancer survivors eventually experience locoregional or metastatic recurrence. It is clear that the adaptive immune system controls proliferative tumor outgrowth, but how dormant tumor cells escape to eventually form new lesions remains unclear. Previous studies showed that proliferative D2A1 or D2.OR tumors are restricted by adaptive immunity in Balb/c versus SCID mice whereas dormant D2.1 tumors are largely unaffected. Further, D2.1 tumors are highly infiltrated with T cells, particularly CD4+ cells and Tregs. Thus, the aim of the present study was to determine the extent to which dormant D2.1 tumors evade antigen-specific immunity and how these tumors shape the T cell niche. Experiments were performed using Balb/c syngeneic D2 mammary tumor cells – D2A1 cells grow rapidly upon orthotopic injection, D2.OR cells proliferate but to a lesser degree than D2A1, and D2.1 cells remain dormant long term. Cells were also modified to express enhanced green fluorescent protein (eGFP) for use with Just eGFP Death Inducing (JeDI) T cells containing a T cell receptor (TCR) targeted to eGFP200-208. Transfer of JeDI T cells caused complete rejection of D2A1-eGFP tumors in the mammary fat pad (MFP) while no difference was observed in D2.1-eGFP tumors. In contrast, treatment with anti-CTLA4 alone, anti-PD-1 alone, or combined anti-CTLA4/PD-1 reduced D2.1 tumor progression compared to isotype control. Whole JeDI splenocytes were also stimulated in vitro with eGFP200-208 peptide in the presence of D2A1 or D2.1 conditioned medium (CM). Surprisingly, while CD8 cells proliferated in both D2A1 and D2.1 CM, CD4+ T cells also proliferated in D2.1 CM and the Treg fraction of CD4+ cells was significantly elevated in D2.1 CM compared to D2A1 CM or control medium. RNA-sequencing confirmed substantial immune activity in D2.1 tumors compared to D2A1. D2.1 tumors also displayed a hybrid epithelial/mesenchymal (E/M) phenotype and expressed genes associated with both mammary epithelium (Krt8, Krt14, Cdh1) and mesenchymal cells (Itga5, Snai2, Notch1, Zeb1). Moreover, Gene Set Enrichment Analysis (GSEA) showed an enrichment of mammary stem and luminal progenitor cell signatures in D2.1 tumors. Of the top upregulated genes, Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) was chosen for further investigation. Interestingly, increased CD8 proliferation and IFNγ production, but decreased CD4 and Treg abundance, was observed in DKK3 shRNA knockdown (k/d) D2.1 CM compared to shScramble control. In vivo, overexpressing DKK3 in D2A1 cells significantly increased MFP tumor growth in Balb/c but not SCID mice, altered T cell activation locally and systemically, and decreased eGFP-specific antibodies. Similar results were observed in DKK3-overexpressing D2.OR tumors. Conversely, DKK3 k/d in D2.1 cells resulted in ~60% complete tumor rejection. Together, these data indicate that CD4 cells, particularly Tregs, are crucial in establishing a protective niche for long-term tumor dormancy with tumor-derived DKK3 emerging as a mediator of the Treg niche. Citation Format: Timothy N. Trotter, Delila Serra, Carina Dagotto, Tao Wang, Xiao Yang, Junping Wei, Gangjun Lei, H. Kim Lyerly, Zachary C. Hartman. Dormant mammary tumors persist long-term despite adaptive immunity by establishing a Treg-dominated niche via DKK3 [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR015.

Multiple myeloma (MM) is a plasma-cell malignancy that grows in bone marrow (BM). MM resistance to chemotherapy is a major cause of patient mortality. Osteocytes are the most abundant cell type in bone. We have recently demonstrated that MM cells induce osteocyte apoptosis. While the mechanisms responsible for MM-induced osteocyte apoptosis have been described, it is unknown whether apoptotic osteocytes provide feedback that regulates MM cells. To study this, we developed a unique syngenic animal model of murine MM in which osteocyte apoptosis can be induced through targeted expression of human diphtheria toxin receptor (DTR) in osteocytes and subsequent injection of DT. In our current study, we used this DTR mouse model to access the impact of osteocyte apoptosis on MM cell response to the treatments of bortezomib (BTZ) and dexamethasone (DEX), two frontline anti-MM drugs. Eight-week-old DTR mice were i.p. injected with either PBS as control or DT (12.5µg/kg) to induce apoptosis in osteocytes. One week later, DTR+PBS and DTR+DT mice were injected with 106 5TGM1-luc MM cells via tail vein (n=21/group) and after 4 days, randomly assigned to treatment for 4 weeks with PBS or DEX (i.p. 1mg/kg/day) or BTZ (i.p. 0.5mg/kg/3 days) (n=7/group). Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1-luc MM cells) ELISA, performed after 4-week treatment, demonstrated that osteocyte apoptosis promotes tumor growth in BM; DEX or BTZ treatment significantly inhibited tumor growth in DTR+PBS mice but not in DTR+DT mice. Flow cytometry analysis of 5TGM1-Luc MM cells in the BM further revealed that DEX and BTZ failed to induce MM cell apoptosis/death in DTR+DT mice, in contrast to the effectiveness observed in DTR+PBS mice. Analyses of BM cells harvested from these mice showed that compared with tumor-bearing DTR+PBS+PBS mice, tumor-bearing DTR+DT+PBS mice had significantly higher concentration of immunosuppressive myeloid-derived suppressor cells (MDSCs) and fewer effective CD8+ T cells in the BM. These CD8+ T cells also have a lower expression of the cytotoxic molecules granzyme B and IFN-γ. Importantly, DEX or BTZ treatment improved anti-MM immunity in the BM of tumor-bearing DTR+PBS mice but failed in tumor-bearing OB-Runx2-/- mice. Gemcitabine (GEM) is a FDA-approved anti-cancer agent and MDSC inhibitor used in the treatment of solid tumors but not commonly used in MM therapy. To confirm that MDSCs indeed promote osteocyte-apoptosis-induced MM resistance to DEX/BTZ and to determine if depletion of MDSCs can overcome this resistance, we treated 5TGM1-Luc tumor-bearing DTR+DT mice with PBS, GEM (i.p. 30 mg/kg/week), GEM+DEX or GEM+BTZ for 4 weeks. Bioluminescence imaging and serum IgG2bκ ELISA demonstrated that GEM+DEX or GEM+BTZ reversed MM resistance to DEX and BTZ and resulted in the greatest reduction in tumor burden among all treatments. In conclusion, our findings demonstrate that osteocyte apoptosis promotes MM cell resistance to DEX and BTZ and BM immunosuppression induced by osteocyte apoptosis plays a role in this event. Citation Format: Xiaoxiao Hao, Chao Zhang, Timothy N Trotter, Pramod S Gowda, Yang Yang. Osteocyte apoptosis induces an immunosuppressive microenvironment in bone marrow and promotes myeloma chemoresistance [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A12.

Two HER2-specific mAbs, trastuzumab and pertuzumab (T+P), combined with chemotherapy comprise standard-of-care treatment for advanced HER2+ breast cancers (BC). While this antibody combination is highly effective, its synergistic mechanism-of-action (MOA) remains incompletely understood. Past studies have suggested that the synergy underlying this combination occurs through the different mechanisms elicited by these antibodies, with pertuzumab suppressing HER2 heterodimerization and trastuzumab inducing antitumor immunity. However, in vivo evidence for this synergy is lacking. In this study, we found that the therapeutic efficacy elicited by their combination occurs through their joint ability to activate the classical complement pathway, resulting in both complement-dependent cytotoxicity and complement-dependent cellular phagocytosis of HER2+ tumors. We also demonstrate that tumor C1q expression is positively associated with survival outcome in HER2+ BC patients and that complement regulators CD55 and CD59 were inversely correlated with outcome, suggesting the clinical importance of complement activity. Accordingly, inhibition of C1q in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant antitumor MOA for T+P therapy that may be functionally enhanced to potentially augment clinical therapeutic efficacy.

Little is known regarding why the immune system is ineffective at eliminating certain disseminated/dormant tumor cells before overt metastatic recurrence. Previous findings revealed that the adaptive immune system reduces growth of proliferative D2A1 tumors in Balb/c versus SCID mice while dormant D2.1 tumors are largely unaffected by adaptive immunity, despite being significantly infiltrated by T cells. Therefore, the aim of the present study was to determine the downstream effects and molecular underpinnings of dormancy-mediated alterations to the immune environment. Experiments were performed using two independent clones of a spontaneous mammary tumor from a female Balb/c mouse that display varying degrees of dormancy in vivo. D2A1 cells grow rapidly upon orthotopic or systemic injection while D2.1 cells remain dormant long term in local and metastatic sites. Both cell lines were modified to express enhanced green fluorescent protein (eGFP) for use with Just eGFP Death Inducing (JeDI) T cells which contain a T cell receptor (TCR) targeted to eGFP200-208. Adoptive transfer of JeDI T cells caused complete rejection of D2A1-eGFP tumors in the mammary fat pad (MFP), while no difference in growth was observed in D2.1-eGFP tumors. Interestingly, contralateral MFP implantation of D2.1-eGFP tumors resulted in a partial rescue of D2A1-eGFP rejection suggesting systemic immune suppression by distant, dormant tumor cells. Bulk RNA-sequencing of D2A1 and D2.1 tumors revealed significant upregulation of pathways associated with immune activation and extracellular matrix (ECM) remodeling in dormant tumors. Further, Gene Set Enrichment Analysis (GSEA) showed an enrichment for signatures of mammary stem cells and luminal progenitor cells, as well as exhausted vs memory CD8 cells and T cell anergy, in D2.1 tumors. Of the top upregulated genes in D2.1 tumors, pleiotrophin (PTN) and Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) were chosen for further investigation. Overexpressing PTN or DKK3 in D2A1 cells significantly increased MFP tumor growth in Balb/c but not SCID mice. In addition, PTN or DKK3 overexpression altered both local and systemic T cell profiles, including decreased effector memory (CD62l-, CD44+) and increased naïve (CD62l+, CD44-) CD4 and CD8 cells. Humoral immunity was also affected, as indicated by decreased eGFP-specific antibodies in serum from mice bearing PTN or DKK3 overexpressing tumors. Conversely, knocking down PTN or DKK3 in D2.1 cells had the opposite effect on systemic immunity. Taken together, these data indicate that dormant tumors can resist antigen-specific killing by CD8 T cells despite high T cell infiltration, and possibly suppress antigen-specific immunity in distant tumor sites. Moreover, both PTN and DKK3 emerge as potential mediators of dormancy-induced immune evasion. Citation Format: Timothy N. Trotter, Delila Serra, Carina Dagotto, Tao Wang, Xiao Yang, Junping Wei, Gangjun Lei, H. Kim Lyerly, Zachary C. Hartman. Dormant mammary tumors resist antigen-specific killing and regulate systemic immunity in association with PTN and DKK3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2451.

Background: Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM). Although the proteasome inhibitor bortezomib (BTZ) is one of the most effective front-line chemotherapeutic drugs for MM, 15–20% of high-risk patients do not respond to this drug or become resistant to treatment. The mechanisms driving this chemoresistance remain unclear. Previous studies showed that the tumor microenvironment contributes to cancer chemoresistance. Our recent studies demonstrated that Runt-related transcription factor 2 (Runx2) deficiency in osteoblasts (OBs) creates a cytokine-rich and immunosuppressive microenvironment in the BM and promotes MM progression. However, the impact of Runx2 deficiency in OBs on the efficacy of BTZ in treatment of MM is still unknown. Methods: We assessed the effects of OB-Runx2 deficiency on the outcome of BTZ treatment in OB-Runx2 +/+ and OB-Runx2 -/- mouse models with MM using bioluminescence imaging, ELISA and flow cytometry. In addition, we used a co-culturing in vitro system to explore the mechanism of BTZ resistance and assessed this system by MTT assays and Western blot analysis. Results: We discovered that OB-Runx2 deficiency induces MM cells resistance to BTZ via the suppression of immunity and increased active TGF-β1 in the BM. We further demonstrated that depletion of myeloid-derived suppressor cells (MDSCs) by gemcitabine or inhibition of TGF-ß1 activity by SRI31277, a compound that blocks thrombospondin 1 (TSP1)-mediated TGF-ß1 activation, restores anti-tumor immunity in the BM and overcomes BTZ resistance induced by OB-Runx2 deficiency. In addition, SRI31277 also directly inhibits the activity of canonical (Smad2/3) and non-canonical (Erk1/2) signaling pathways of TGF-ß1 in 5TGM1-Luc MM cells and sensitizes MM cells to BTZ, resulting in increased apoptosis of MM cells. Conclusions: OB-Runx2 deficiency promotes BTZ resistance in MM cells through the regulations of MDSCs and TSP1-mediated TGF-ß1 activation in the BM. These data identify novel mechanisms of BTZ resistance in MM and suggest new strategies to overcome BTZ resistance in treatment of MM.

In multiple myeloma, abnormal plasma cells accumulate and proliferate in the bone marrow. Recently, we observed that Runx2, a bone-specific transcription factor, is highly expressed in multiple myeloma cells and is a major driver of multiple myeloma progression in bone. The primary goal of the present study was to identify Runx2-targeting miRNAs that can reduce tumor growth. Expression analysis of a panel of miRNAs in multiple myeloma patient specimens, compared with healthy control specimens, revealed that metastatic multiple myeloma cells express low levels of miR-342 and miR-363 but high levels of Runx2. Reconstituting multiple myeloma cells (CAG) with miR-342 and miR-363 reduced the abundance of Runx2 and the expression of metastasis-promoting Runx2 target genes RANKL and DKK1, and suppressed Runx2 downstream signaling pathways Akt/β-catenin/survivin, which are required for multiple myeloma tumor progression. Intravenous injection of multiple myeloma cells (5TGM1), stably overexpressing miR-342 and miR-363 alone or together, into syngeneic C57Bl/KaLwRij mice resulted in a significant suppression of 5TGM1 cell growth, decreased osteoclasts and increased osteoblasts, and increased antitumor immunity in the bone marrow, compared with mice injected with 5TGM1 cells expressing a miR-Scramble control. In summary, these results demonstrate that enhanced expression of miR-342 and miR-363 in multiple myeloma cells inhibits Runx2 expression and multiple myeloma growth, decreases osteolysis, and enhances antitumor immunity. Thus, restoring the function of Runx2-targeting by miR-342 and miR-363 in multiple myeloma cells may afford a therapeutic benefit by preventing multiple myeloma progression. Implications: miR-342 and miR-363–mediated downregulation of Runx2 expression in multiple myeloma cells prevents multiple myeloma progression. Mol Cancer Res; 16(7); 1138–48. ©2018 AACR.

Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib (BTZ) is one of the most effective front-line chemotherapeutic drugs for MM; however, 15-20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that MM cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OBs). In the current study, we investigated the impact of OB-Runx2 deficiency on the outcome of BTZ treatment using our syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 -/- mice) and OB-Runx2 +/+ mice as control. Five-week-old OB-Runx2 +/+ mice and OB-Runx2 -/- mice were i.v. injected with 5TGM1-Luc murine MM cells (2×10 6). On day 8, after the tumor injection, mice were randomly assigned to treatment for 4 weeks, with either BTZ (i.p. injection, 0.5 mg/kg body weight, twice/week) or PBS. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA showed that BTZ significantly inhibited tumor growth in OB-Runx2 +/+ mice, but not in OB-Runx2 -/- mice. Cytokine array and ELISA showed that in PBS-treated, tumor-bearing OB-Runx2 -/- mice, BM levels of thrombospondin-1 (TSP-1), a matricellular protein that converts latent TGF-β1 to its active form, and active TGF-β1 were significantly higher than levels in tumor-bearing OB-Runx2 +/+ counterparts. Interestingly, BTZ treatment further increased the levels of both TSP-1 and active TGF-β1 in the BM of tumor-bearing OB-Runx2 -/- mice, but it did not affect the level of either TSP-1 or TGF-β1 in the BM of tumor-bearing OB-Runx2 +/+ mice. These results suggest that OB-Runx2 deficiency increases TGF-β1 activation via TSP-1 in BM and BTZ treatment further enhances this effect. SRI31277 is a tripeptide antagonist that blocks TSP-1-mediated activation of TGF-β1 and it has been shown to reduce tumor burden, TGF-β signaling, and osteolytic bone disease in multiple mouse models of MM. To confirm the involvement of TSP-1/TGF-β1 activation in MM BTZ resistance induced by OB-Runx2 deficiency and to test whether blocking TSP1-mediated TGF-β1 activation can alleviate BTZ resistance, 5TGM1-Luc MM tumor-bearing OB-Runx2 +/+ and OB-Runx2 -/- mice were treated with PBS, BTZ, SRI31277 (osmotic pump, 30 mg/kg body weight per day), or BTZ + SRI31277 for 4 weeks. While SRI31277 did not further increase BTZ-induced MM inhibition in OB-Runx2 +/+ mice, SRI31277 treatment significantly reduced the tumor burden in OB-Runx2 -/- mice. Furthermore, treatment with BTZ + SRI31277 augmented the reduction in tumor burden induced by SRI31277 treatment alone. Flow cytometry and Western blot analyses demonstrated that SRI31277 treatment enhanced apoptosis, reduced Ki-67 expression as well as the activity of both canonical (SMAD2/3) and non-canonical (ERK1/2) signaling pathways of TGF-β1 in MM cells in OB-Runx2 -/- mice; and BTZ+SRI31277 treatment augmented these effects. Furthermore, TGF-β has immunosuppressive effects in MM and our data show that SRI31277 overcomes this by reducing myeloid derived suppressor cells, checkpoint, and T cell exhaustion markers and by increasing cytotoxic T cells. In conclusion, OB-Runx2 deficiency, induced by MM cells, promotes MM resistance to BTZ through the upregulation of TGF-β1 activation in the BM and TGF-β1 signaling in MM cells. Importantly, blocking TSP-1-mediated TGF-β1 activation with SRI31277 can reverse this resistance and immune dysfunction. Our findings demonstrate a novel mechanism for BTZ resistance in MM and identify a new target and strategy for overcoming immune dysregulation and chemoresistance in MM. Disclosures Murphy-Ullrich: Millipore Sigma: Patents & Royalties.

The HER2-specific monoclonal antibody (mAb), trastuzumab, has been the mainstay of therapy for HER2(+) breast cancer (BC) for approximately 20 years. However, its therapeutic mechanism of action (MOA) remains unclear, with antitumor responses to trastuzumab remaining heterogeneous and metastatic HER2(+) BC remaining incurable. Consequently, understanding its MOA could enable rational strategies to enhance its efficacy. Using both murine and human versions of trastuzumab, we found its antitumor activity dependent on Fcγ receptor stimulation of tumor-associated macrophages (TAMs) and antibody-dependent cellular phagocytosis (ADCP), but not cellular cytotoxicity (ADCC). Trastuzumab also stimulated TAM activation and expansion, but did not require adaptive immunity, natural killer cells, and/or neutrophils. Moreover, inhibition of the innate immune ADCP checkpoint, CD47, significantly enhanced trastuzumab-mediated ADCP and TAM expansion and activation, resulting in the emergence of a unique hyperphagocytic macrophage population, improved antitumor responses, and prolonged survival. In addition, we found that tumor-associated CD47 expression was inversely associated with survival in HER2(+) BC patients and that human HER2(+) BC xenografts treated with trastuzumab plus CD47 inhibition underwent complete tumor regression. Collectively, our study identifies trastuzumab-mediated ADCP as an important antitumor MOA that may be clinically enabled by CD47 blockade to augment therapeutic efficacy.

Multiple myeloma (MM) cells reside in the bone marrow microenvironment and form complicated interactions with nonneoplastic, resident stromal cells. We previously found that aggressive MM cells shift osteoblast progenitors toward adipogenesis. In addition, adipocytes are among the most common cell types in the adult skeleton; both mature adipocytes and preadipocytes serve as endocrine cells that secrete a number of soluble molecules into the microenvironment. Therefore, we used a combination of in vivo and in vitro methods to test the hypothesis that an increase in adipocyte lineage cells feeds back to promote MM progression. The results of this study revealed that bone marrow from patients with MM indeed contains increased preadipocytes and significantly larger mature adipocytes than normal bone marrow. We also found that preadipocytes and mature adipocytes secrete many molecules important for supporting MM cells in the bone marrow and directly recruit MM cells through both monocyte chemotactic protein-1 and stromal cell-derived factor-1α. Co-culture experiments found that preadipocytes activate Wnt signaling and decrease cleaved caspase-3, whereas mature adipocytes activate ERK signaling in MM cells. Furthermore, mature adipocyte conditioned medium promotes MM growth, whereas co-culture with preadipocytes results in enhanced MM cell chemotaxis in vitro and increased tumor growth in bone in vivo . Combined, these data reveal the importance of preadipocytes and mature adipocytes on MM progression and represent a unique target in the bone marrow microenvironment.

Metastasis is the major cause of the death in cancer patients, and a frequent site of metastasis for many cancers is the bone marrow. Therefore, understanding the mechanisms underlying the metastatic process is necessary for future prevention and treatment. The tumor microenvironment is now known to play a role in the metastatic cascade, both at the primary tumor and in metastatic sites, and includes both cellular and non-cellular components. The extracellular matrix (ECM) provides structural support and signaling cues to cells. One particular group of molecules associated with the ECM, known as matricellular proteins, modulate multiple aspects of tumor biology, including growth, migration, invasion, angiogenesis and metastasis. These proteins are also important for normal function in the bone by regulating bone formation and bone resorption. Recent studies have described a link between some of these proteins and metastasis of various tumors to the bone. The aim of this review is to summarize what is currently known about matricellular protein influence on bone metastasis. Particular attention to the contribution of both tumor cells and non-malignant cells in the bone has been given.

Many cancer survivors respond to early treatment with complete remission only to succumb to the disease after the tumor returns, sometimes decades later, in a distant metastatic site. Tumor cells can exist in a nonproliferative state of dormancy (quiescence) in specific, perivascular niches in the lungs, brain, and especially bone marrow. In particular, dormant breast tumors can recur after long periods of time, even over 15 years after treatment of the primary tumor. Why the immune system is ineffective at eliminating disseminated/dormant tumor cells before growing into new metastatic lesions is currently unknown. Therefore, in the current study we hypothesized that dormant tumor cells actively elicit a reduced adaptive immune response compared to proliferative tumor cells, ultimately aiding in long-term survival and persistence of dormant cells. We utilized the established syngeneic D2 series of cell lines, which comprises independent clones from a spontaneous mammary tumor in a female Balb/c mouse with varying degrees of dormancy—D2A1 cells grow rapidly upon injection while D2.1 remain dormant. Mammary fat pad (MFP) injections (n=8/group) revealed that proliferative D2A1 tumors rapidly grew in the absence of an adaptive immune system in SCID mice and reached terminal endpoints after 2 to 3 weeks. In comparison, D2A1 tumor growth was significantly reduced in syngeneic Balb/c mice (p Citation Format: Timothy N. Trotter, Cong-Xiao Liu, Tao Wang, H. Kim Lyerly, Zachary C. Hartman. Dormant breast tumor cells avoid the adaptive immune system through regulation of the immune and tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B107.

Bone dissemination and bone disease occur in approximately 80% of patients with multiple myeloma (MM) and are a major cause of patient mortality. We previously demonstrated that MM cell-derived heparanase (HPSE) is a major driver of MM dissemination to and progression in new bone sites. However the mechanism(s) by which HPSE promotes MM progression remains unclear. In the present study, we investigated the involvement of mesenchymal features in HPSE-promoted MM progression in bone. Using a combination of molecular, biochemical, cellular, and in vivo approaches, we demonstrated that (1) HPSE enhanced the expression of mesenchymal markers in both MM and vascular endothelial cells; (2) HPSE expression in patient myeloma cells positively correlated with the expression of the mesenchymal markers vimentin and fibronectin. Additional mechanistic studies revealed that the enhanced mesenchymal-like phenotype induced by HPSE in MM cells is due, at least in part, to the stimulation of the ERK signaling pathway. Finally, knockdown of vimentin in HPSE expressing MM cells resulted in significantly attenuated MM cell dissemination and tumor growth in vivo. Collectively, these data demonstrate that the mesenchymal features induced by HPSE in MM cells contribute to enhanced tumor cell motility and bone-dissemination.

A major cause of morbidity in patients with multiple myeloma is the development and progression of bone disease. Myeloma bone disease is characterized by rampant osteolysis in the presence of absent or diminished bone formation. Heparanase, an enzyme that acts both at the cell-surface and within the extracellular matrix to degrade polymeric heparan sulfate chains, is upregulated in a variety of human cancers including multiple myeloma. We and others have shown that heparanase enhances osteoclastogenesis and bone loss. However, increased osteolysis is only one element of the spectrum of myeloma bone disease. In the present study, we hypothesized that heparanase would also affect mesenchymal cells in the bone microenvironment and investigated the effect of heparanase on the differentiation of osteoblast/stromal lineage cells. Using a combination of molecular, biochemical, cellular and in vivo approaches, we demonstrated that heparanase significantly inhibited osteoblast differentiation and mineralization, and reduced bone formation in vivo. In addition, heparanase shifts the differentiation potential of osteoblast progenitors from osteoblastogenesis to adipogenesis. Mechanistically, this shift in cell fate is due, at least in part, to heparanase-enhanced production and secretion of the Wnt signaling pathway inhibitor DKK1 by both osteoblast progenitors and myeloma cells. Collectively, these data provide important new insights into the role of heparanase in all aspects of myeloma bone disease and strongly support the use of heparanase inhibitors in the treatment of multiple myeloma.

The total synthesis of 7-deoxy-6-O-methylfusarentin (1) and a formal synthesis of 7-deoxy-6,8-O-dimethylfusarentin (2) has been successfully achieved in 10 steps. The described tactic underscores a diastereoselective strategy which incorporates a single acyclic reaction based on the initial stereocenter by means of a 1,3-chelation-controlled Reetz-Keck-type allylation.

The progression of multiple myeloma (MM) is governed by a network of molecular signals, the majority of which remain to be identified. Recent studies suggest that Runt-related transcription factor 2 (Runx2), a well-known bone-specific transcription factor, is also expressed in solid tumors, where expression promotes both bone metastasis and osteolysis. However, the function of Runx2 in MM remains unknown. The current study demonstrated that (1) Runx2 expression in primary human MM cells is significantly greater than in plasma cells from healthy donors and patients with monoclonal gammopathy of undetermined significance; (2) high levels of Runx2 expression in MM cells are associated with a high-risk population of MM patients; and (3) overexpression of Runx2 in MM cells enhanced tumor growth and disease progression in vivo. Additional studies demonstrated that MM cell–derived Runx2 promotes tumor progression through a mechanism involving the upregulation of Akt/β-catenin/Survivin signaling and enhanced expression of multiple metastatic genes/proteins, as well as the induction of a bone-resident cell-like phenotype in MM cells. Thus, Runx2 expression supports the aggressive phenotype of MM and is correlated with poor prognosis. These data implicate Runx2 expression as a major regulator of MM progression in bone and myeloma bone disease.

In Multiple myeloma (MM), abnormal plasma cells accumulate in the bone marrow and spread to new bone sites. However, the mechanisms underlying this spread of MM cells remain unclear. Runx2 is a bone specific transcription factor upregulated in various human tumors, including MM. Our studies have previously demonstrated that Runx2 is a major driver of MM progression in bone. In the present study, our goal was to identify miRNAs targeting Runx2 to reduce tumor growth and dissemination of MM to new bone sites and asses their therapeutic potential. Expression analysis of a panel of miRNA's regulating Runx2 revealed an inverse relationship between Runx2 expression and two miRNAs: miR-342 and miR-363. miRNA expression analysis using the Gene Expression Omnibus database showed that miR-342 and miR-363 are highly expressed in plasma cells of normal donors (n= 3), while Runx2 is detected at very low levels. In contrast, MM cells of patients with newly diagnosed (n=23) and relapsed MM (n=17) expressed very little miR-342 and miR-363 but showed high levels of Runx2 expression. Reconstituting CAG human MM cells with synthetic miR-342, miR-363 or miR-342+miR-363 in combination reduced the expression of Runx2 and the metastasis-promoting Runx2 target genes RANKL, DKK1 and DMP1. Tumor cell viability and migration were also decreased compared to control in vitro. We then transfected miR-342, miR-363 or miR-342+miR-363 in mouse 5TGM1 MM cells expressing high levels of Runx2 and tested bone-homing and growth in syngenic C57Bl/KaLwRij mice by intravenous injection. Mouse sera were collected at week 2 and 4 after tumor cell injection and the levels of IgG2bκ, a marker of 5TGM1 cells, were measured by ELISA. The results showed significantly decreased IgG2bκ levels in mice bearing miR-342 or miR-363 transfected tumors compared to mice bearing control miR-scramble tumors. Additionally, miR-342+miR-363 co-transfection produced a synergistic effect on reducing tumor growth. Mechanistic studies demonstrated that miR-342 and miR-363 induced Runx2 suppression, results in inhibition of Runx2-downstream signaling pathways Akt/β-catenin/survivin, which are required for MM tumor progression. In conclusion, we have identified two novel miRNAs, miR-342 and miR-363, that negatively regulate Runx2 expression in MM cells. We further demonstrated that enhanced expression of miR-342 or miR-363 in MM cells inhibits MM growth in vivo. Thus, miR-342 and miR-363 are potential novel markers of MM prognosis and can be developed as new therapeutic drugs for MM treatment. Citation Format: Pramod S. Gowda, Mohammad Q. Hassan, Timothy N. Trotter, Yang Yang. Targeting myeloma cell-derived runx2 by miRNAs suppresses multiple myeloma growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2525. doi:10.1158/1538-7445.AM2017-2525

Multiple Myeloma (MM) pathogenesis and progression is intricately tied to the bone marrow (BM) microenvironment, and understanding this microenvironment is of upmost importance. Osteocytes are the most abundant cell type in bone, comprising over 90% of the cellular population. These cells were long thought to be primarily inert mechanical sensors, however osteocyte regulation of the BM is increasingly appreciated. Still, very little is known about the role of osteocytes in the context of cancer. We initially evaluated MM patient BM biopsies and determined that osteocyte apoptosis is increased compared to MGUS and healthy BM. We further validated this finding in the SCID-hu mouse model - increased osteocyte death was seen in the primary human bone grafts that were injected with aggressive MM cells compared to those injected with less aggressive MM cells. Surprisingly, and more interestingly, increased osteocyte apoptosis/death was also found in the contralateral, un-injected human bone grafts from mice with aggressive MM. Importantly, these changes in the secondary bone grafts occurred prior to detection of MM cells. In the present study, we investigated if and how osteocyte apoptosis at distant bone sites, before MM metastasis, feeds back to MM cells to support dissemination and progression. We developed a unique, syngeneic mouse model of MM in which osteocytes can be specifically ablated in vivo. Mice expressing the Diphtheria toxin receptor (DTR) in mature osteocytes (RIKEN BioResource Center, Japan) were crossed with C57Bl/KaLwRij mice, a strain that when injected with syngeneic 5TGM1 MM cells develops many characteristics of human MM. The resulting 8-week old progeny expressing the DTR gene and on the C57Bl/KaLwRij background were intraperitoneally (i.p.) injected with Diphtheria toxin (DT,12.5 μg/kg) or PBS as control (n=7 per group). After 7 days, mice were injected intravenously (i.v.) with 2x106 5TGM1 MM cells expressing firefly Luciferase. Weekly bioluminescent imaging and ELISA for IgG2bκ (secreted by 5TGM1 cells) tracked tumor location and growth over time. Remarkably, compared to control mice (DTR+PBS), osteocyte ablated (DTR+DT) mice showed significantly enhanced tumor homing to bone by bioluminescent imaging and significantly increased total tumor burden by both bioluminescent imaging and sera IgG2bκ ELISA. To understand mechanistically how osteocyte apoptosis enhances MM-bone homing and tumor progression, we evaluated BM microenvironmental changes after osteocyte apoptosis was induced. Syngeneic DTR mice were injected at 8 weeks with DT or PBS (n=3-4 per group). Mouse legs were collected after 7 days: one leg for sectioning and one leg for isolating BM cells and BM extracellular fluid. Flow cytometry was performed on BM cells and BM extracellular fluid was evaluated using a cytokine/chemokine array. Leg sections were stained via immunohistochemistry (IHC). Cytokine arrays of BM extracellular fluid revealed substantial increases of multiple cytokines/chemokines important for MM dissemination and progression (i.e. IL-6, HGF) in DTR+DT mice. Additionally, bone destruction-associated molecules DKK1, RANKL, and M-CSF were also increased in DTR + DT mice compared to DTR+PBS control mice. IHC showed increased numbers of osteocytes positive for sclerostin, HIF-1α and RANKL. Finally, flow cytometric analysis suggested heightened immune suppression in the BM as multiple immune-suppressive cell populations were enhanced in DTR+DT mice compared to DTR+PBS mice. These include myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and a regulatory B cell subset (Breg). In conclusion, we have successfully generated a syngeneic model of MM in which osteocytes are selectively ablated in vivo after a single injection of DT. Using this model, our data demonstrate that osteocyte apoptosis supports MM homing and growth in bone, likely as a result of an altered BM microenvironment. Interestingly, these changes include enhanced MM supportive/attractive soluble molecules, support of bone destruction, and enhanced immune suppression. Combined, our data suggests that osteocyte apoptosis is important for developing a pre-metastatic niche and contributes to MM progression in bone. Therefore, preventing osteocyte apoptosis may help stall and prevent dissemination of MM, and possibly other cancers, to bone. Disclosures No relevant conflicts of interest to declare.

Background: Multiple myeloma (MM) is hematologic malignancy of plasma cells that thrives in and progresses throughout the bone marrow microenvironment. The bone marrow is host to a variety of cell types, including bone marrow stromal cells and hematopoietic cells, as well as osteoblasts, osteoclasts and adipocytes. We and others have shown that MM cells not only alter the local bone microenvironment to support MM progression, but also modify distant bone sites through secretion of soluble factors before arrival of tumor cells. One critical alteration in bone is the shift of osteoblast progenitor cells from osteoblastogenesis towards adipogenesis. Whether and how these increased adipocyte lineage cells contribute to MM cells homing to and growth in bone are currently unknown. Both mature and pre-adipocytes have multiple endocrine functions such as cytokine and growth factor secretion. Thus, an increase in adipocyte lineage cells likely alters the bone microenvironment in favor of supporting MM. Here, we hypothesized that adipocytes and their precursors play an active role in MM progression that contributes to MM growth and dissemination throughout bone. Methods: Our hypothesis was tested using a co-culture system in which 3T3-L1 mouse pre-adipocytes or mature adipocytes were separated by a porous membrane from 5TGM1-luc mouse MM cells. This system allowed cross-talk by secreted molecules but not through direct cell-cell contact. After three days of co-culture, MM cells were collected for (i) intravenous (I.V.) tail-vein injections into syngeneic C57Bl/KaLwRiJ mice or (ii) protein collection for Western blot analyses. For in vivo experiments, tumor progression was tracked by bioluminescent luciferase imaging and total tumor burden was evaluated by IgG2bκ (a soluble marker of 5TGM1-luc MM cells) levels in mouse serum. In addition, conditioned medium (CM) was collected from either pre-adipocytes or mature adipocytes for MM cell migration assays or for analyses of soluble factors in the CM by cytokine/chemokine array. Results: I.V. injection of 5TGM1-luc MM cells into mice revealed that those previously co-cultured with pre-adipocytes more rapidly homed to bone and grew larger tumors compared to 5TGM1-luc MM cells cultured alone, whereas the MM cells cultured with mature adipocytes showed no significant increase in either bone homing or growth. Analysis of pre-adipocyte and mature adipocyte CM by cytokine/chemokine arrays demonstrated that pre-adipocytes secrete significantly more HGF, MCP-1, OPN and SDF-1α compared to mature adipocytes. Migration assays in which pre-adipocyte or mature adipocyte CM was used as a chemoattractant indicated that MM cells migrate significantly more towards both pre-adipocyte and mature adipocyte CM than fresh media. However, the pre-adipocyte CM exhibited significantly more chemoattraction than mature adipocyte CM. Furthermore, addition of an MCP-1 or SDF-1α neutralizing antibody to both pre-adipocyte CM and mature adipocyte CM resulted in significantly reduced migration of MM cells. However, pre-adipocyte CM required higher concentrations of antibodies than mature adipocyte CM, indicating a higher concentration of MCP-1 and SDF-1α in pre-adipocyte CM. MM cells also exhibited a significant dose-dependent migration towards recombinant pre-adipocyte factor-1 (Pref-1), a marker of pre-adipocytes that is down-regulated during adipogenesis. Finally, Western blots revealed that co-culture of MM cells with pre-adipocytes resulted in activation of β-catenin signaling, which is important for cell proliferation, survival and motility, in MM cells. Conclusions: These data indicate that adipocyte lineage cells play active but differentiation-dependent roles in MM progression, likely via the secretion of soluble factors. Both pre-adipocytes and mature adipocytes directly attract MM cells by secreting chemoattractants such as MCP-1 and SDF-1α. Interestingly, our data identify pre-adipocytes, and not mature adipocytes, as the main driver of the aggressive bone phenotype of MM cells. In sum, these data suggest that an increase in adipocyte lineage cells in the bone marrow at distant bone sites could feed back to MM cells and support MM dissemination to these distant bone sites. Studies to determine the intricacies of this novel role of pre-adipocytes in MM are currently ongoing. Disclosures Suva: University of Arkansas for Medical Sciences: Employment.

Multiple myeloma (MM), a hematologic malignancy, preferentially grows in bone marrow and frequently metastasizes between bones. Our previous studies have demonstrated that heparanase (HPSE), an enzyme overexpressed in MM cells, promotes MM bone-metastasis and progression. However, the mechanism of HPSE action is unclear. Epithelial-mesenchymal-transition (EMT) is a process giving epithelial cells the features of mesenchymal cells which has been shown to be critical in the metastatic process of epithelial-origin tumors. Although MM is not an epithelial-derived malignancy, MM cells indeed express epithelial marker E-cadherin and mesenchymal marker Vimentin, and have the ability to adhere to and interact with the extracellular matrix (ECM). In the present study, we determined the role of EMT in HPSE-induced MM bone metastasis via: (1) Using Western blot to evaluate the expression of EMT markers E-cadherin, Vimentin and Fibronectin in MM cells that were transfected with empty vector (HPSE-low) or HPSE cDNA (HPSE-high), as well as MM cells treated with recombinant human HPSE (rHPSE) for 48hrs; (2) staining HPSE and EMT markers on tumors established by HPSE-low or HPSE-high cells in SCID mice (n = 10), and on bone marrow biopsies of 35 MM patients; (3) knocking down Vimentin in HPSE-high-luc MM cells by Vimentin shRNA (VIM k/d-luc cells) and testing bone homing of VIM k/d-luc cells in SCID mice by i.v. and s.c. injection of these cells. Tumor growth and bone-metastasis was determined by measuring human kappa levels (a soluble marker of MM cells) in mouse sera and weekly bioluminescent imaging. Western blots demonstrated HPSE-high and rHPSE treated MM cells expressed significantly higher levels of the mesenchymal markers Vimentin and Fibronectin, and lower levels of the epithelial marker E-cadherin, compared to HPSE-low or PBS treated MM cells. Significantly increased expression of Vimentin and decreased expression of E-cadherin was revealed in tumors formed by HPSE-high cells compared with the tumors formed by HPSE-low cells. Immunostaining of bone marrow biopsies of MM patients indicated HPSE and Vimentin expression in MM cells have a significant positive correlation (rs = 0.414, p = 0.014). Finally, both SCID-i.v. injection and SCID-s.c. injection models showed that knocking down mesenchymal marker Vimentin in HPSE-high MM cells successfully blocked HPSE-induced bone metastasis and inhibited tumor growth, compared with mice bearing shRNA control HPSE-high MM cells. In conclusion, our studies demonstrated (1) EMT-like features are a novel biomarker of poor prognosis in MM; (2) HPSE promotes MM metastasis to bone and disease progression, at least in part, through inducing EMT-like features in MM cells; (3) Inhibition of Vimentin expression in MM cells impairs HPSE-induced tumor growth and bone metastasis. Note: This abstract was not presented at the meeting. Citation Format: Qianying Pan, Juan Li, Patrick D. Rowan, Timothy N. Trotter, Yang Yang. The involvement of EMT in heparanase promoted bone-metastasis in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2015-4102

Multiple myeloma (MM) is a hematologic malignancy of plasma cells that thrives in and metastasizes throughout the bone marrow microenvironment. This microenvironment is host to a variety of cell types, including osteoblasts, osteoclasts and stromal cells, that become altered both at the primary tumor site and distant bone sites to support the growth and spread of MM cells. We recently reported that under certain conditions, such as when MM cells overexpress heparanase, MM cells redirect osteoblast progenitors from osteoblastogenesis towards adipogenesis. In addition, adipocytes naturally accumulate in aging marrow and adipocytes/adipose tissues have endocrine functions, secreting soluble factors such as adiponectin and leptin. Thus, adipocytes have the potential to be influential on MM cell behavior. Here, we hypothesized that adipocytes play an active role in MM progression through the secretion of soluble molecules that promote MM growth and metastasis to bone. To test this we used a co-culture system in which 3T3-L1 mouse pre-adipocytes or mature adipocytes were separated by a porous membrane from 5TGM1 mouse MM cells, allowing cross-talk by secreted molecules but not direct cell-cell contact. We then tested the motility of these “educated” 5TGM1 MM cells by migration assay. Somewhat surprisingly, we found that it was not mature adipocyte but pre-adipocyte education that enhanced the migration ability of MM cells compared to MM cells cultured alone. In a separate experiment, we injected 5TGM1 MM cells cultured alone, with pre-adipocytes, or with mature adipocytes into syngeneic C57BL/KaLwRij mice via tail vein and monitored progression by bioluminescent imaging. Total tumor burden was evaluated by IgG2bκ (a soluble marker of 5TGM1 MM cells) levels in mouse serum. Compared to MM cells cultured alone, those cultured with pre-adipocytes homed to the bone sooner and grew faster whereas those cultured with mature adipocytes showed no difference in homing or growth. To identify factors that may contribute to this apparent difference, pre-adipocyte or mature adipocyte conditioned media (CM) was analyzed using a cytokines/chemokines array. The results showed that pre-adipocytes secrete significantly larger quantities of molecules HGF, MCP-1 and IL-6 than mature adipocytes. Additional migration assays also confirmed that MM cells migrate more towards pre-adipocyte CM than to fresh media alone or mature adipocyte CM. In conclusion, this study demonstrates that pre-adipocytes, through the secretion of a variety of soluble molecules, promote a more aggressive phenotype in MM cells and contribute to MM growth and bone homing. Citation Format: Timothy N. Trotter, Patrick D. Rowan, Qianying Pan, Yang Yang. Pre-adipocytes promote myeloma homing to and growth in bone by secretion of soluble molecules. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2387. doi:10.1158/1538-7445.AM2015-2387

Background. Multiple myeloma (MM), an incurable plasma-cell malignancy, preferentially grows in bone marrow and frequently metastasizes to new bone sites. The molecular network responsible for progression and bone-metastasis of MM remains largely unknown. Recent work from our laboratory suggests that the Runx2 transcription factor, well-known for its essential role in osteoblast differentiation and bone development, is also required for coordinating the network of molecular signals in MM cells that drive MM progression and bone metastasis. Substantial evidence exists about expression of Runx2 in various cancer cells, including myeloma cells. Moreover, Runx2 is shown to promote tumor-to-bone metastasis, osteolytic bone disease, and tumor progression in solid tumors. However, the role of Runx2 in MM progression and bone metastasis remains unknown. In the present study, we determined the mechanism(s) governing the action of Runx2 in MM cell, especially during tumor growth and bone-homing. Methods. Molecular, biochemical, and in vivo approaches were used to assess the role of Runx2 in growth and homing of MM to bone tissue. Initially, bone marrow biopsies from 35 MM patients and 14 healthy bone marrow donors were examined for the expression of Runx2. For a mechanistic understanding of the role of Runx2 in MM, we over-expressed and knocked-down Runx2 expression in both murine 5TGM1 and human MM1S myeloma cells. The following methods and strategies were used to examine the Runx2 over-expression (Runx2 k/in), Runx2 knockdown (Runx2 k/d) and control MM cell lines in both ex vivo and in vivo models. (1) Assessment of tumor growth and homing in the 5TGM1 syngenic model of murine myeloma (for 5TGM1 cells) and SCID mice (for MM1S cells). (2) RNA-Sequencing, Real-time PCR and Western blot analyses to determine downstream genes/molecules regulated by Runx2 in MM cells. (3) Cytokine array and ELISA of the conditioned media to identify Runx2 regulated soluble factors secreted by MM cells. Results. Immunostaining of bone marrow biopsies indicated that Runx2 expression was significantly increased in both cytoplasm and nuclei of MM cells compared to normal plasma cells (p< 0.001). In vivo studies using three different animal models (i.v., subcutaneous and intra-tibial injection) demonstrated that the overexpression of Runx2 in MM cells promoted tumor growth and homing to bone whereas knockdown of Runx2 inhibited tumor progression and bone-homing. Interestingly, Runx2 over-expression in MM cells leads to the increased expression of active β-catenin and phospho-AKT, indicating that Runx2 promotes tumor growth through Wnt/β-catenin and AKT signaling pathway. This regulatory mechanism was further confirmed in 5TGM1 Runx2 k/d cells, in which both the expression of active β-catenin and phospho-AKT was significantly decreased. RNA-sequencing and real-time PCR revealed that, over-expression of Runx2 induced the expression of multiple marker genes of osteoblast (Osteocalcin, osteopontin), the osteocytes (DMP1) as well as the osteoclast (cathepsin K, RANK). Expression of these marker genes was significantly down-regulated in Runx2 k/d 5TGM1 cells. These results imply that Runx2 promotes tumor bone-homing by providing MM cells with the ability to “mimic” bone marrow resident cells and therefore escape immune surveillance in the host bone marrow. Finally, we provided evidence that MM cell-derived Runx2 promote secretion of multiple soluble factors linked with increased metastasis such as DKK1, MMP9, SDF-1, and osteopontin by MM cells. Conclusions. MM cell-derived Runx2 is a central regulator of MM progression. Runx2 expression promotes MM growth and bone-metastasis via upregulation of (i) Wnt/β-catenin and AKT signaling, (ii) the expression of multiple osteogenic genes, and (iii) the secretion of metastatic soluble factors, in MM cells. This activation could activate a pre-metastatic niche in bone thereby providing MM cells with the ability to avoid immune surveillance in the bone marrow, and supporting survival and growth of tumor cells that arrive in bone. These discoveries provide insights into MM pathology, help to predict the prognosis of MM patients, and identify novel therapeutic targets for MM. Disclosures No relevant conflicts of interest to declare.

Background. Bone metastases occur in 80% of patients with multiple myeloma (MM) and are a major cause of patient mortality. We previously demonstrated that MM cell-derived heparanase (HPSE), an enzyme that cleaves heparan sulfate chains, is a master regulator of MM metastasis to bone. However the mechanism by which HPSE promotes MM cell metastasis remains unclear. Recently, the epithelial-mesenchymal-transition (EMT) of cancer cells, a process which gives epithelial cells the features of mesenchymal cells, was shown to play a critical role in the metastases of solid tumors. This process enables tumor cells to lose cell-cell and cell-extracellular matrix (ECM) adhesion, and acquire motility and invasiveness, thereby facilitating metastasis. Unlike solid tumors, MM is not an epithelial-derived malignancy. MM cells do not have cell junctions, but do adhere to and interact with the ECM. Our study has shown that MM cells express both the epithelial marker E-cadherin and the mesenchymal marker vimentin. However, the existence and potential role of EMT in MM has not been investigated. Thus, in the present study, the involvement of EMT in HPSE-induced MM bone metastasis was investigated. Methods. To assess the involvement of EMT in HPSE-promoted MM growth and metastasis, cellular protein was isolated from HPSE-low (CAG cells transfected with empty vector) and HPSE-high (CAG cells transfected with HPSE cDNA) MM cells, as well as from wild-type CAG and RPMI 8226 MM cells treated with recombinant HPSE (rHPSE) for 48 hours. Western blotting was performed and the expression of EMT markers (Vimentin, Fibronectin and RANK) was determined. The signaling pathway(s) involved in HPSE-induced EMT were identified by culturing HPSE-high cells or RPMI8226 myeloma cells with rHPSE in the presence or absence of specific signaling pathway inhibitors and measuring the expression of EMT markers. To assess the involvement of EMT in HPSE-promoted tumor progression in vivo, HPSE-low or HPSE-high MM cells were injected subcutaneously into SCID mice. Tumor growth was determined by the measurement of human kappa level in mouse sera and bone-metastasis monitored by luciferase imaging. Tumors were harvested 6 weeks after tumor cell injection and stained for EMT marker expression. Finally, to investigate whether HPSE-high MM cells induce an EMT-like phenotype in endothelial blood vessel cells, human umbilical vein endothelial cells (HUVECs) were cultured in conditioned medium of HPSE-low or HPSE-high cells for 48 hrs, and cell lysates were analyzed for EMT marker expression. Results. Western blots demonstrated that HPSE-high MM cells as well as CAG and RPMI 8226 MM cells treated with rHPSE expressed significantly elevated levels of the mesenchymal markers Vimentin, Fibronectin and RANK, and lower levels of the epithelial marker E-cadherin, compared to HPSE-low or the untreated cells, indicating HPSE induced EMT in MM cells. The enhanced EMT in HPSE-high or rHPSE-treated MM cells was reversed by the addition of the potent ERK inhibitor PD98059, suggesting the direct involvement of ERK signaling in HPSE-induced EMT. In vivo, tumor burden and bone metastasis was significantly enhanced in mice bearing HPSE-high tumors compared with mice bearing HPSE-low tumors. Significantly higher expression of Vimentin, Fibronectin and RANK and decreased expression of E-cadherin was revealed in HPSE-high tumors, compared with the tumors formed by HPSE-low cells. In addition, the conditioned medium from HPSE-high cells significantly increased Vimentin and decreased VE-cadherin expression in HUVECs. Therefore, the same EMT in vascular endothelial cells can be induced by soluble factors secreted from HPSE-expressing MM cells in vitro. Conclusions. (1) HPSE promotes MM progression and metastasis by inducing EMT-like features in MM cells via stimulation of the ERK signaling pathway. (2) MM cell-derived HPSE indirectly induces EMT of vascular endothelial cells. We propose that the EMT-like features induced by HPSE in both MM cells and vascular endothelial cells equips the cells with higher mobility and invasiveness, contributing to the enhanced angiogenesis and tumor bone-metastasis commonly found in MM. Disclosures No relevant conflicts of interest to declare.