Lysophosphatidic acid modulates CD8 T cell immunosurveillance and metabolism to impair anti-tumor immunity.

Lysophosphatidic acid (LPA) is a bioactive lipid which increases in concentration locally and systemically across different cancer types. Yet, the exact mechanism(s) of how LPA affects CD8 T cell immunosurveillance during tumor progression remain unknown. We show LPA receptor (LPAR) signaling by CD8 T cells promotes tolerogenic states via metabolic reprogramming and potentiating exhaustive-like differentiation to modulate anti-tumor immunity. We found LPA levels predict response to immunotherapy and Lpar5 signaling promotes cellular states associated with exhausted phenotypes on CD8 T cells. Importantly, we show that Lpar5 regulates CD8 T cell respiration, proton leak, and reactive oxygen species. Together, our findings reveal that LPA serves as a lipid-regulated immune checkpoint by modulating metabolic efficiency through LPAR5 signaling on CD8 T cells. Our study offers key insights into the mechanisms governing adaptive anti-tumor immunity and demonstrates LPA could be exploited as a T cell directed therapy to improve dysfunctional anti-tumor immunity.

Targeting CDK4/6 Represents a Therapeutic Vulnerability in Acquired BRAF/MEK Inhibitor-Resistant Melanoma.

There is a clear need to identify targetable drivers of resistance and potential biomarkers for salvage therapy for patients with melanoma refractory to the combination of BRAF and MEK inhibition. In this study, we performed whole-exome sequencing on BRAF-V600E-mutant melanoma patient tumors refractory to the combination of BRAF/MEK inhibition and identified acquired oncogenic mutations in NRAS and loss of the tumor suppressor gene CDKN2A We hypothesized the acquired resistance mechanisms to BRAF/MEK inhibition were reactivation of the MAPK pathway and activation of the cell-cycle pathway, which can both be targeted pharmacologically with the combination of a MEK inhibitor (trametinib) and a CDK4/6 inhibitor (palbociclib). In vivo, we found that combination of CDK4/6 and MEK inhibition significantly decreased tumor growth in two BRAF/MEK inhibitor-resistant patient-derived xenograft models. In vitro, we observed that the combination of CDK4/6 and MEK inhibition resulted in synergy and significantly reduced cellular growth, promoted cell-cycle arrest, and effectively inhibited downstream signaling of MAPK and cell-cycle pathways in BRAF inhibitor-resistant cell lines. Knockdown of CDKN2A in BRAF inhibitor-resistant cells increased sensitivity to CDK4/6 inhibition alone and in combination with MEK inhibition. A key implication of our study is that the combination of CDK4/6 and MEK inhibitors overcomes acquired resistance to BRAF/MEK inhibitors, and loss of CDKN2A may represent a biomarker of response to the combination. Inhibition of the cell-cycle and MAPK pathway represents a promising strategy for patients with metastatic melanoma who are refractory to BRAF/MEK inhibitor therapy.

Melanoma Metastases to the Adrenal Gland Are Highly Resistant to Immune Checkpoint Inhibitors.

BACKGROUND: Adrenal gland metastases (AGMs) are common in advanced-stage melanoma, occurring in up to 50% of patients. The introduction of immune checkpoint inhibitors (ICIs) has markedly altered the outcome of patients with melanoma. However, despite significant successes, anecdotal evidence has suggested that treatment responses in AGMs are significantly lower than in other metastatic sites. We sought to investigate whether having an AGM is associated with altered outcomes and whether ICI responses are dampened in the adrenal glands. PATIENTS AND METHODS: We retrospectively compared ICI responses and overall survival (OS) in 68 patients with melanoma who were diagnosed with an AGM and a control group of 100 patients without AGMs at a single institution. Response was determined using RECIST 1.1. OS was calculated from time of ICI initiation, anti-PD-1 initiation, initial melanoma diagnosis, and stage IV disease diagnosis. Tumor-infiltrating immune cells were characterized in 9 resected AGMs using immunohistochemical analysis. RESULTS: Response rates of AGMs were significantly lower compared with other metastatic sites in patients with AGMs (16% vs 22%) and compared with those without AGMs (55%). Patients with AGMs also had significantly lower median OS compared with those without AGMs (3.1 years vs not reached, respectively). We further observed that despite this, AGMs exhibited high levels of tumor-infiltrating immune cells. CONCLUSIONS: In this cohort of patients with melanoma, those diagnosed with an AGM had lower ICI response rates and OS. These results suggest that tissue-specific microenvironments of AGMs present unique challenges that may require novel, adrenal gland-directed therapies or surgical resection.

Studying Immunotherapy Resistance in a Melanoma Autologous Humanized Mouse Xenograft.

Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood-derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME). When patient tumors were implanted on aHM, mHM, and NOD/SCID/IL2rg-/- (NSG) cohorts, tumors appeared earlier and grew faster on NSG and mHM cohorts. We observed that immune cells differentiating in the aHM were relatively more capable of circulating peripherally, invading into tumors and interacting with the TME. A heterologous, human leukocyte antigen (HLA-A) matched cohort also yielded slower growing tumors than non-HLA-matched mHM, indicating that a less permissive immune environment inhibits tumor progression. When the aHM, mHM, and NSG cohorts were treated with immunotherapies mirroring what the originating patients received, tumor growth in the aHM accelerated, similar to the progression observed in the patients. This rapid growth was associated with decreased immune cell infiltration, reduced interferon gamma (IFNγ)-related gene expression, and a reduction in STAT3 phosphorylation, events that were replicated in vitro using tumor-derived cell lines. IMPLICATIONS: Engrafted adult HSPCs give rise to more tumor infiltrative immune cells, increased HLA matching leads to slower tumor initiation and growth, and continuing immunotherapy past progression can paradoxically lead to increased growth.

Cancer Cell CD44 Mediates Macrophage/Monocyte-Driven Regulation of Head and Neck Cancer Stem Cells.

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.

Inhibiting Translation Elongation with SVC112 Suppresses Cancer Stem Cells and Inhibits Growth in Head and Neck Squamous Carcinoma.

Cancer stem cells (CSC) drive growth, therapy resistance, and recurrence in head and neck squamous cell carcinoma (HNSCC). Regulation of protein translation is crucial for normal stem cells and CSCs; its inhibition could disrupt stemness properties, but translation inhibitors are limited clinically due to toxicity. SVC112 is a synthetic derivative of bouvardin, a plant-derived translation elongation inhibitor. SVC112 had greater antiproliferative effects on HNSCC cells compared with the FDA-approved translation inhibitor omacetaxine mepesuccinate (HHT). SVC112 preferentially inhibited cancer cells compared with patient-matched cancer-associated fibroblasts, whereas HHT was equally toxic to both. SVC112 reduced sphere formation by cell lines and CSCs. SVC112 alone inhibited the growth of patient-derived xenografts (PDX), and SVC112 combined with radiation resulted in tumor regression in HPV-positive and HPV-negative HNSCC PDXs. Notably, CSC depletion after SVC112 correlated with tumor response. SVC112 preferentially impeded ribosomal processing of mRNAs critical for stress response and decreased CSC-related proteins including Myc and Sox2. SVC112 increased cell-cycle progression delay and slowed DNA repair following radiation, enhancing colony and sphere formation radiation effects. In summary, these data demonstrate that SVC112 suppresses CSC-related proteins, enhances the effects of radiation, and blocks growth of HNSCC PDXs by inhibiting CSCs. SIGNIFICANCE: Inhibiting protein elongation with SVC112 reduces tumor growth in head and neck squamous cell carcinoma and increases the effects of radiation by targeting the cancer stem cell pool.

Leading edge or tumor core: Intratumor cancer stem cell niches in oral cavity squamous cell carcinoma and their association with stem cell function.

OBJECTIVES: To describe differences in cancer stem cell (CSC) presence and behavior associated with their intratumor compartment of origin using a patient-derived xenograft (PDX) model of oral cavity squamous cell carcinoma (OCSCC). MATERIALS AND METHODS: Four HPV-negative OCSCC PDX cases were selected (CUHN004, CUHN013, CUHN096, CUHN111) and the percentage of CSCs (ALDH+CD44high) was measured in the tumor Leading Edge (LE) and Core compartments of each PDX tumor case via fluorescence activated cell sorting (FACS). The fraction of cells in the proliferative phase was measured by Ki-67 labelling index of paraffin embedded tissue. The proliferation and invasion of LE versus Core CSCs were compared using sphere and Matrigel invasion assays, respectively. RESULTS: Both CUHN111 and CUHN004 demonstrate CSC enrichment in their LE compartments while CUHN013 and CUHN096 show no intratumor difference. Cases with LE CSC enrichment demonstrate greater Ki-67 labelling at the LE. CSC proliferative potential, assessed by sphere formation, reveals greater sphere formation in CUHN111 LE CSCs, but no difference between CUHN013 LE and Core CSCs. CUHN111 CSCs do not demonstrate an intratumor difference in invasiveness while CUHN013 LE CSCs are more invasive than Core CSCs. CONCLUSION: A discrete intratumor CSC niche is present in a subset of OCSCC PDX tumors. The CSC functional phenotype with regard to proliferation and invasion is associated with the intratumor compartment of origin of the CSC: LE or Core. These individual functional characteristics appear to be modulated independently of one another and independently of the presence of an intratumor CSC niche.

Wnt signaling dynamics in head and neck squamous cell cancer tumor-stroma interactions.

Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.

BRAF fusions identified in melanomas have variable treatment responses and phenotypes.

Oncogenic BRAF fusions have emerged as an alternate mechanism for BRAF activation in melanomas and other cancers. A number of BRAF fusions with different 5' gene partners and BRAF exon breakpoints have been described, but the effects of different partners and breakpoints on cancer phenotypes and treatment responses has not been well characterized. Targeted RNA sequencing was used to screen 60 melanoma patient-derived xenograft (PDX) models for BRAF fusions. We identified three unique BRAF fusions, including a novel SEPT3-BRAF fusion, occurring in four tumors (4/60, 6.7%), all of which were "pan-negative" (lacking other common mutations) (4/18, 22.2%). The BRAF fusion PDX models showed variable growth rates and responses to MAPK inhibitors in vivo. Overexpression of BRAF fusions identified in our study, as well as other BRAF fusions previously identified in melanomas, resulted in a high degree of variability in 2D proliferation and 3D invasion between the different fusions. While exogenously expressed BRAF fusions all responded to MAPK inhibition in vitro, we observed potential differences in signaling and feedback mechanisms. In summary, BRAF fusions are actionable therapeutic targets, however there are significant differences in phenotypes, treatment responses, and signaling which may be clinically relevant.

Dual use of hematopoietic and mesenchymal stem cells enhances engraftment and immune cell trafficking in an allogeneic humanized mouse model of head and neck cancer.

In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.

Salivary Gland Cancer Patient-Derived Xenografts Enable Characterization of Cancer Stem Cells and New Gene Events Associated with Tumor Progression.

Purpose: Salivary gland cancers (SGC) frequently present with distant metastases many years after diagnosis, suggesting a cancer stem cell (CSC) subpopulation that initiates late recurrences; however, current models are limited both in their availability and suitability to characterize these rare cells.Experimental Design: Patient-derived xenografts (PDX) were generated by engrafting patient tissue onto nude mice from one acinic cell carcinoma (AciCC), four adenoid cystic carcinoma (ACC), and three mucoepidermoid carcinoma (MEC) cases, which were derived from successive relapses from the same MEC patient. Patient and PDX samples were analyzed by RNA-seq and Exome-seq. Sphere formation potential and in vivo tumorigenicity was assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations.Results: For successive MEC relapses we found a time-dependent increase in CSCs (ALDH+CD44high), increasing from 0.2% to 4.5% (P=0.033), but more importantly we observed an increase in individual CSC sphere formation and tumorigenic potential. A 50% increase in mutational burden was documented in subsequent MEC tumors, and this was associated with increased expression of tumor-promoting genes (MT1E, LGR5, and LEF1), decreased expression of tumor-suppressor genes (CDKN2B, SIK1, and TP53), and higher expression of CSC-related proteins such as SOX2, MYC, and ALDH1A1. Finally, genomic analyses identified a novel NFIB-MTFR2 fusion in an ACC tumor and confirmed previously reported fusions (NTRK3-ETV6 and MYB-NFIB)Conclusions: Sequential MEC PDX models preserved key patient features and enabled the identification of genetic events putatively contributing to increases in both CSC proportion and intrinsic tumorigenicity, which mirrored the patient's clinical course. Clin Cancer Res; 24(12); 2935-43. ©2018 AACR.