Purpose: Patients with relapsed pediatric solid malignancies have few therapeutic options, and many of these patients die of their disease. B7-H3 is an immune checkpoint protein encoded by the CD276 gene that is overexpressed in many pediatric cancers. Here, we investigate the activity of the B7-H3–targeting antibody–drug conjugate (ADC) m276-SL-PBD in pediatric solid malignancy patient-derived (PDX) and cell line–derived xenograft (CDX) models. Experimental Design: B7-H3 expression was quantified by RNA sequencing and by IHC on pediatric PDX microarrays. We tested the safety and efficacy of m276-SL-PBD in two stages. Randomized trials of m276-SL-PBD of 0.5 mg/kg on days 1, 8, and 15 compared with vehicle were performed in PDX or CDX models of Ewing sarcoma (N = 3), rhabdomyosarcoma (N = 4), Wilms tumors (N = 2), osteosarcoma (N = 5), and neuroblastoma (N = 12). We then performed a single mouse trial in 47 PDX or CDX models using a single 0.5 m/kg dose of m276-SL-PBD. Results: The vast majority of PDX and CDX samples studied showed intense membranous B7-H3 expression (median H-score 177, SD 52). In the randomized trials, m276-SL-PBD showed a 92.3% response rate, with 61.5% of models showing a maintained complete response (MCR). These data were confirmed in the single mouse trial with an overall response rate of 91.5% and MCR rate of 64.4%. Treatment-related mortality rate was 5.5% with late weight loss observed in a subset of models dosed once a week for 3 weeks. Conclusions: m276-SL-PBD has significant antitumor activity across a broad panel of pediatric solid tumor PDX models.
Ewing sarcoma is the second most common pediatric bone cancer, with a 5-year survival rate for metastatic disease of only 20%. Recent work indicates that survival is strongly correlated with high levels of tumor-infiltrating lymphocytes (TIL), whose abundance is associated with IFN-inducible chemokines CXCL10 and CCL5. However, the tumor-intrinsic factors that drive chemokine production and TIL recruitment have not been fully elucidated. We previously showed that ubiquitin-specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in Ewing sarcoma cells. Here, we show that this gene set comprises chemokines associated with immunostimulatory, antitumorigenic functions, including CXCL10 and CCL5. USP6 synergistically enhanced chemokine production in response to exogenous IFN by inducing surface upregulation of IFNAR1 and IFNGR1. USP6-expressing Ewing sarcoma cells stimulated migration of primary human monocytes and T lymphocytes and triggered activation of natural killer (NK) cells in vitro. USP6 inhibited Ewing sarcoma xenograft growth in nude but not NSG mice and was accompanied by increased intratumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the antitumorigenic effects of USP6. High USP6 expression in patients with Ewing sarcoma was associated with chemokine production, immune infiltration, and improved survival. This work reveals a previously unrecognized tumor-suppressive function for USP6, which engenders an immunostimulatory microenvironment through pleiotropic effects on multiple immune lineages. This further raises the possibility that USP6 activity may be harnessed to create a "hot" tumor microenvironment in immunotherapy.Significance: This study reveals a novel tumor-suppressive function for USP6 by inducing an immunostimulatory microenvironment, suggesting that USP6 activity may be exploited to enhance immunotherapy regimens.
<div>AbstractPurpose:<p>Patients with relapsed pediatric solid malignancies have few therapeutic options, and many of these patients die of their disease. B7-H3 is an immune checkpoint protein encoded by the <i>CD276</i> gene that is overexpressed in many pediatric cancers. Here, we investigate the activity of the B7-H3–targeting antibody–drug conjugate (ADC) m276-SL-PBD in pediatric solid malignancy patient-derived (PDX) and cell line–derived xenograft (CDX) models.</p>Experimental Design:<p>B7-H3 expression was quantified by RNA sequencing and by IHC on pediatric PDX microarrays. We tested the safety and efficacy of m276-SL-PBD in two stages. Randomized trials of m276-SL-PBD of 0.5 mg/kg on days 1, 8, and 15 compared with vehicle were performed in PDX or CDX models of Ewing sarcoma (<i>N</i> = 3), rhabdomyosarcoma (<i>N</i> = 4), Wilms tumors (<i>N</i> = 2), osteosarcoma (<i>N</i> = 5), and neuroblastoma (<i>N</i> = 12). We then performed a single mouse trial in 47 PDX or CDX models using a single 0.5 m/kg dose of m276-SL-PBD.</p>Results:<p>The vast majority of PDX and CDX samples studied showed intense membranous B7-H3 expression (median H-score 177, SD 52). In the randomized trials, m276-SL-PBD showed a 92.3% response rate, with 61.5% of models showing a maintained complete response (MCR). These data were confirmed in the single mouse trial with an overall response rate of 91.5% and MCR rate of 64.4%. Treatment-related mortality rate was 5.5% with late weight loss observed in a subset of models dosed once a week for 3 weeks.</p>Conclusions:<p>m276-SL-PBD has significant antitumor activity across a broad panel of pediatric solid tumor PDX models.</p></div>
BACKGROUND: Neuroblastoma is a cancer arising from the developing sympathetic nervous system. Less than half of children diagnosed with high-risk neuroblastoma survive five years. Two transcriptional subpopulations have been identified: aggressive, chemotherapy-sensitive adrenergic (ADRN) and indolent, chemotherapy-resistant mesenchymal (MES). ALCAM is a cell adhesion molecule that is overexpressed in neuroblastoma. ALCAM is a cancer stem cell marker in colorectal and small cell lung cancer and promotes bone metastasis in prostate cancer. ALCAM also interacts with the CD6 receptor on T cells, which facilitates adhesion in the immunological synapse and inhibits T cell activation. METHODS: We characterized ALCAM expression in 24 neuroblastoma cell lines using immunoblotting. We investigated ALCAM expression in ADRN and MES neuroblastoma using RNA-sequencing data from two patient datasets. Next, we developed doxycycline (dox)-inducible ALCAM depletion cell lines using CRISPR inhibition (CRISPRi) in three high-ALCAM cell lines with relevant oncogenic aberrations. Using the Incucyte® SX5 Live-Cell Analysis System, we interrogated the effects of ALCAM depletion on proliferation and scratch wound migration. To determine the mechanism of ALCAM overexpression, we leveraged ChIP-sequencing data for MYC and MYCN in 9 cell lines. We probed the effects of the MYC(N)-MAX disrupter MYCi975 on ALCAM expression using immunoblotting. To investigate the effects of the ALCAM-CD6 interaction on T cells, we stimulated CD4+ and CD8+ T cells with CD3/28 antibodies in the presence or absence of recombinant ALCAM and measured activation by ELISA for IFN-γ, and TNF-α and flow cytometry for CD69 expression. RESULTS: ALCAM is highly expressed in most neuroblastoma primary tumors and cell lines. ALCAM mRNA expression is higher in the ADRN subpopulation (p ≤ 0.0045), although both ADRN and MES tumors and cell lines express high levels of ALCAM. Dox-inducible CRISPRi mediated robust ALCAM depletion in all three cell lines, and ALCAM knockdown significantly delayed proliferation (p ≤ 0.0025) and impaired scratch wound migration. ChIP-sequencing showed robust MYC(N) peaks at the ALCAM promoter, and treatment with IC50 concentrations (3.5 - 9.0 μM) of MYCi975 depleted MYC(N) and ALCAM. Activation of CD4+ and CD8+ T cells in the presence of recombinant ALCAM mediated dose-dependent reduction in expression of T cell activation marker CD69 after 3 days and inhibited IFN-γ and TNF-α release. CONCLUSIONS: ALCAM is overexpressed in neuroblastoma and its expression is driven, at least in part, by MYC(N). ALCAM is a mediator of cellular proliferation and migration and may contribute to immune evasion by inhibiting T cell activation. Ongoing in vivo studies will characterize the effects of ALCAM depletion on tumor growth and metastasis. We propose ALCAM is a tractable immunotherapy target through multi-specific antibodies or the ALCAM-CD6 interaction. Citation Format: Jarrett Lindsay, Minu Samanta, Nathan Kendsersky, Jonathan Gaither, Molly Christie, Kyabeth Torres-Rodriguez, Catherine Wingrove, John M. Maris. ALCAM promotes neuroblastoma proliferation, migration, and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6714.
<div>Abstract<p>Ewing sarcoma is the second most common pediatric bone cancer, with a 5-year survival rate for metastatic disease of only 20%. Recent work indicates that survival is strongly correlated with high levels of tumor-infiltrating lymphocytes (TIL), whose abundance is associated with IFN-inducible chemokines CXCL10 and CCL5. However, the tumor-intrinsic factors that drive chemokine production and TIL recruitment have not been fully elucidated. We previously showed that ubiquitin-specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in Ewing sarcoma cells. Here, we show that this gene set comprises chemokines associated with immunostimulatory, antitumorigenic functions, including CXCL10 and CCL5. USP6 synergistically enhanced chemokine production in response to exogenous IFN by inducing surface upregulation of IFNAR1 and IFNGR1. USP6-expressing Ewing sarcoma cells stimulated migration of primary human monocytes and T lymphocytes and triggered activation of natural killer (NK) cells <i>in vitro</i>. USP6 inhibited Ewing sarcoma xenograft growth in nude but not NSG mice and was accompanied by increased intratumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the antitumorigenic effects of USP6. High USP6 expression in patients with Ewing sarcoma was associated with chemokine production, immune infiltration, and improved survival. This work reveals a previously unrecognized tumor-suppressive function for USP6, which engenders an immunostimulatory microenvironment through pleiotropic effects on multiple immune lineages. This further raises the possibility that USP6 activity may be harnessed to create a “hot” tumor microenvironment in immunotherapy.</p>Significance:<p>This study reveals a novel tumor-suppressive function for USP6 by inducing an immunostimulatory microenvironment, suggesting that USP6 activity may be exploited to enhance immunotherapy regimens.</p></div>
<p>Supplemental Tables 1-2 and Supplemental Figures 1-10</p>
BACKGROUND High-risk neuroblastoma is a pediatric cancer arising from the developing sympathetic nervous system with a 50% relapse rate that is typically fatal. At least two subpopulations of neuroblastoma cells exist that can transdifferentiate, adrenergic and mesenchymal, the latter being more resistant to chemotherapy. Mechanisms of therapy resistance are largely unknown and the cells responsible for relapse have not been identified. METHODS We used single nucleus RNA and ATAC sequencing to identify and characterize the cells that survive chemotherapy, termed here “persister cells”, from a cohort of 20 matched diagnostic and post induction chemotherapyhigh-risk neuroblastoma patients and two patient derived xenograft (PDX) models from diagnostic tumors. Eight representative cell lines derived from neuroblastomas at diagnosis were treated with standard-of-care chemotherapy, and flow cytometry was used to sort for live cells. ML120B and CRISPR-CAS9 were used to modulate NF-kB signaling. An RNA-seq dataset of 153 high-risk neuroblastoma patients was used to determine differentially activated pathways between adrenergic and mesenchymal tumors. RESULTS Residual malignant cells in the post-chemotherapy tumor samples clustered into three main groups separated by the response to therapy. The most prevalent group of persister cells in responders (N=16/20) displayed low MYC(N) activity even in the presence of MYCN amplification. This group also demonstrated decreased expression of the adrenergic core regulatory circuit genes including PHOX2B, ISL1, HAND2, along with marked activation of TNF-alpha via NF-kB signaling. High NF-kB activity was found in a subpopulation of diagnostic cells in two chemo-refractory patients. We validated decreased expression of MYCN (2-fold decrease, p<0.0001) and PHOX2B (3.13-fold decrease, p<0.0001) in PDXs following chemotherapy. MYCN protein levels were decreased and nuclear p65 levels were increased in cell lines treated with chemotherapy. Pharmacologic inhibition of NF-kB signaling and genetic depletion of p65 resulted in increased killing (3.58-fold increase, p=0.0012) of neuroblastoma cell lines in response to chemotherapy. Finally, we classified 153 diagnostic high-risk neuroblastomas as predominantly adrenergic or mesenchymal using RNA-seq, showing that mesenchymal tumors were enriched with NF-kB pathway activation signatures. We then validated high nuclear p65 levels in 3 mesenchymal cell lines. We tested 6 adrenergic lines, 4 of which had no detectable nuclear p65. Notably, the 2 cell lines with detectable nuclear p65 were derived from diagnostic specimens that showed de novo chemotherapy resistance. CONCLUSIONS NF-kB activation is a major mediator of de novo and acquired chemotherapy resistance in high-risk neuroblastoma. We postulate that concomitant silencing of this pathway could eliminate persister cells and prevent disease relapse. Citation Format: Liron D. Grossmann, Yasin Uzun, Jarrett Lindsay, Chia-Hui Chen, Catherine Wingrove, Peng Gao, Anusha Thadi, Quinlen Marshall, Nathan M. Kendsersky, Lea Surrey, Daniel Martinez, Emily Mycek, Colleen Casey, Kateryna Krytska, Matthew Tsang, Adam Wolpaw, David N. Groff, Erin Runbeck, Jayne McDevitt, Dinh Diep, Tasleema Patel, Kathrin M. Bernt, Chi Dang, Kun Zhang, Yael P. Mosse, Kai Tan, John M. Maris. NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma [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 699.
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