PAK4 inhibition improves PD1 blockade immunotherapy in prostate cancer by increasing immune infiltration.

Antitumor immunity requires lymphocytes to localize to the tumor. Prostate cancers (PCs) are immunologically cold and tend to lack T-cell infiltration. Most advanced PCs are insensitive to PD1 blockade therapies. Using syngeneic RM1 prostate tumors, p21-activated kinase-4 (PAK4) knockdown (KD) and pharmacological inhibition was assessed in C57BL/6J mice treated with PD1 antibodies (αPD1). RNASeq was used to characterize the immune response in the tumor. Immunohistochemistry, flow cytometry, and in vivo blocking studies confirmed the role of cell surface proteins in the generation of immune responses. In The Cancer Genome Atlas, PAK4 expression was inversely correlated with immune cell infiltration. PAK4 expression was controlled by the androgen receptor and its pioneering factor, FOXA1. PAK4 KD increased CD8+ T-cell infiltration and expression of IFNγ response genes. PAK4 KD also upregulated angiogenesis and endothelial cell adhesion molecules in the tumor microenvironment, contributing to CD8+ lymphocyte recruitment. Pharmacological inhibition of PAK4 made PC more responsive to immunotherapy with αPD1. A decrease in PAK4 activity increases immune activation and vascularity, which increases CD8+ lymphocyte infiltration into the tumor. Therefore, targeting PAK4 may improve the response of human PC to immunotherapy.

Melanoma dedifferentiation induced by IFN-γ epigenetic remodeling in response to anti-PD-1 therapy.

Melanoma dedifferentiation has been reported to be a state of cellular resistance to targeted therapies and immunotherapies as cancer cells revert to a more primitive cellular phenotype. Here, we show that, counterintuitively, the biopsies of patient tumors that responded to anti-programmed cell death 1 (anti-PD-1) therapy had decreased expression of melanocytic markers and increased neural crest markers, suggesting treatment-induced dedifferentiation. When modeling the effects in vitro, we documented that melanoma cell lines that were originally differentiated underwent a process of neural crest dedifferentiation when continuously exposed to IFN-γ, through global chromatin landscape changes that led to enrichment in specific hyperaccessible chromatin regions. The IFN-γ-induced dedifferentiation signature corresponded with improved outcomes in patients with melanoma, challenging the notion that neural crest dedifferentiation is entirely an adverse phenotype.

Conserved Interferon-γ Signaling Drives Clinical Response to Immune Checkpoint Blockade Therapy in Melanoma.

We analyze the transcriptome of baseline and on-therapy tumor biopsies from 101 patients with advanced melanoma treated with nivolumab (anti-PD-1) alone or combined with ipilimumab (anti-CTLA-4). We find that T cell infiltration and interferon-γ (IFN-γ) signaling signatures correspond most highly with clinical response to therapy, with a reciprocal decrease in cell-cycle and WNT signaling pathways in responding biopsies. We model the interaction in 58 human cell lines, where IFN-γ in vitro exposure leads to a conserved transcriptome response unless cells have IFN-γ receptor alterations. This conserved IFN-γ transcriptome response in melanoma cells serves to amplify the antitumor immune response. Therefore, the magnitude of the antitumor T cell response and the corresponding downstream IFN-γ signaling are the main drivers of clinical response or resistance to immune checkpoint blockade therapy.

Landscape of somatic single nucleotide variants and indels in colorectal cancer and impact on survival.

Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR = 0.42, 95% CI: 0.21-0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR = 1.53, 95% CI: 1.21-1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features.

Deep Functional and Molecular Characterization of a High-Risk Undifferentiated Pleomorphic Sarcoma.

Nonrhabdomyosarcoma soft-tissue sarcomas (STSs) are a class of 50+ cancers arising in muscle and soft tissues of children, adolescents, and adults. Rarity of each subtype often precludes subtype-specific preclinical research, leaving many STS patients with limited treatment options should frontline therapy be insufficient. When clinical options are exhausted, personalized therapy assignment approaches may help direct patient care. Here, we report the results of an adult female STS patient with relapsed undifferentiated pleomorphic sarcoma (UPS) who self-drove exploration of a wide array of personalized Clinical Laboratory Improvement Amendments (CLIAs) level and research-level diagnostics, including state of the art genomic, proteomic, ex vivo live cell chemosensitivity testing, a patient-derived xenograft model, and immunoscoring. Her therapeutic choices were also diverse, including neoadjuvant chemotherapy, radiation therapy, and surgeries. Adjuvant and recurrence strategies included off-label and natural medicines, several immunotherapies, and N-of-1 approaches. Identified treatment options, especially those validated during the in vivo study, were not introduced into the course of clinical treatment but did provide plausible treatment regimens based on FDA-approved clinical agents.

Overcoming Genetically Based Resistance Mechanisms to PD-1 Blockade.

Mechanism-based strategies to overcome resistance to PD-1 blockade therapy are urgently needed. We developed genetic acquired resistant models of JAK1, JAK2, and B2M loss-of-function mutations by gene knockout in human and murine cell lines. Human melanoma cell lines with JAK1/2 knockout became insensitive to IFN-induced antitumor effects, while B2M knockout was no longer recognized by antigen-specific T cells and hence was resistant to cytotoxicity. All of these mutations led to resistance to anti-PD-1 therapy in vivo. JAK1/2-knockout resistance could be overcome with the activation of innate and adaptive immunity by intratumoral Toll-like receptor 9 agonist administration together with anti-PD-1, mediated by natural killer (NK) and CD8 T cells. B2M-knockout resistance could be overcome by NK-cell and CD4 T-cell activation using the CD122 preferential IL2 agonist bempegaldesleukin. Therefore, mechanistically designed combination therapies can overcome genetic resistance to PD-1 blockade therapy. SIGNIFICANCE: The activation of IFN signaling through pattern recognition receptors and the stimulation of NK cells overcome genetic mechanisms of resistance to PD-1 blockade therapy mediated through deficient IFN receptor and antigen presentation pathways. These approaches are being tested in the clinic to improve the antitumor activity of PD-1 blockade therapy.This article is highlighted in the In This Issue feature, p. 1079.

Chromosomal instability in untreated primary prostate cancer as an indicator of metastatic potential.

BACKGROUND: Metastatic prostate cancer (PC) is highly lethal. The ability to identify primary tumors capable of dissemination is an unmet need in the quest to understand lethal biology and improve patient outcomes. Previous studies have linked chromosomal instability (CIN), which generates aneuploidy following chromosomal missegregation during mitosis, to PC progression. Evidence of CIN includes broad copy number alterations (CNAs) spanning > 300 base pairs of DNA, which may also be measured via RNA expression signatures associated with CNA frequency. Signatures of CIN in metastatic PC, however, have not been interrogated or well defined. We examined a published 70-gene CIN signature (CIN70) in untreated and castration-resistant prostate cancer (CRPC) cohorts from The Cancer Genome Atlas (TCGA) and previously published reports. We also performed transcriptome and CNA analysis in a unique cohort of untreated primary tumors collected from diagnostic prostate needle biopsies (PNBX) of localized (M0) and metastatic (M1) cases to determine if CIN was linked to clinical stage and outcome. METHODS: PNBX were collected from 99 patients treated in the VA Greater Los Angeles (GLA-VA) Healthcare System between 2000 and 2016. Total RNA was extracted from high-grade cancer areas in PNBX cores, followed by RNA sequencing and/or copy number analysis using OncoScan. Multivariate logistic regression analyses permitted calculation of odds ratios for CIN status (high versus low) in an expanded GLA-VA PNBX cohort (n = 121). RESULTS: The CIN70 signature was significantly enriched in primary tumors and CRPC metastases from M1 PC cases. An intersection of gene signatures comprised of differentially expressed genes (DEGs) generated through comparison of M1 versus M0 PNBX and primary CRPC tumors versus metastases revealed a 157-gene "metastasis" signature that was further distilled to 7-genes (PC-CIN) regulating centrosomes, chromosomal segregation, and mitotic spindle assembly. High PC-CIN scores correlated with CRPC, PC-death and all-cause mortality in the expanded GLA-VA PNBX cohort. Interestingly, approximately 1/3 of M1 PNBX cases exhibited low CIN, illuminating differential pathways of lethal PC progression. CONCLUSIONS: Measuring CIN in PNBX by transcriptome profiling is feasible, and the PC-CIN signature may identify patients with a high risk of lethal progression at the time of diagnosis.

Persistence of adoptively transferred T cells with a kinetically engineered IL-2 receptor agonist.

Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities. NKTR-214 is a kinetically-engineered IL-2 receptor ßγ (IL-2Rßγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rßγ. We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model. The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial. In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells.

Copy number alterations are associated with metastatic-lethal progression in prostate cancer.

BACKGROUNDS: Aside from Gleason score few factors accurately identify the subset of prostate cancer (PCa) patients at high risk for metastatic progression. We hypothesized that copy number alterations (CNAs), assessed using CpG methylation probes on Illumina Infinium® Human Methylation450 (HM450K) BeadChip arrays, could identify primary prostate tumors with potential to develop metastatic progression. METHODS: Epigenome-wide DNA methylation profiling was performed in surgically resected primary tumor tissues from two cohorts of PCa patients with clinically localized disease who underwent radical prostatectomy (RP) as primary therapy and were followed prospectively for at least 5 years: (1) a Fred Hutchinson (FH) Cancer Research Center-based cohort (n = 323 patients); and (2) an Eastern Virginia (EV) Medical School-based cohort (n = 78 patients). CNAs were identified using the R package ChAMP. Metastasis was confirmed by positive bone scan, MRI, CT or biopsy, and death certificates confirmed cause of death. RESULTS: We detected 15 recurrent CNAs were associated with metastasis in the FH cohort and replicated in the EV cohort (p < 0.05) without adjusting for Gleason score in the model. Eleven of the recurrent CNAs were associated with metastatic progression in the FH cohort and validated in the EV cohort (p < 0.05) when adjusting for Gleason score. CONCLUSIONS: This study shows that CNAs can be reliably detected from HM450K-based DNA methylation data. There are 11 recurrent CNAs showing association with metastatic-lethal events following RP and improving prediction over Gleason score. Genes affected by these CNAs may functionally relate to tumor aggressiveness and metastatic progression.

PAK4 inhibition improves PD-1 blockade immunotherapy.

Lack of tumor infiltration by immune cells is the main mechanism of primary resistance to programmed cell death protein 1 (PD-1) blockade therapies for cancer. It has been postulated that cancer cell-intrinsic mechanisms may actively exclude T cells from tumors, suggesting that the finding of actionable molecules that could be inhibited to increase T cell infiltration may synergize with checkpoint inhibitor immunotherapy. Here, we show that p21-activated kinase 4 (PAK4) is enriched in non-responding tumor biopsies with low T cell and dendritic cell infiltration. In mouse models, genetic deletion of PAK4 increased T cell infiltration and reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, combination of anti-PD-1 with the PAK4 inhibitor KPT-9274 improved anti-tumor response compared with anti-PD-1 alone. Therefore, high PAK4 expression is correlated with low T cell and dendritic cell infiltration and a lack of response to PD-1 blockade, which could be reversed with PAK4 inhibition.

Publisher Correction: Combined BRAF and MEK inhibition with PD-1 blockade immunotherapy in BRAF-mutant melanoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combined BRAF and MEK inhibition with PD-1 blockade immunotherapy in BRAF-mutant melanoma.

Oncogene-targeted therapy with B-Raf proto-oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors induces a high initial response rate in patients with BRAFV600-mutated melanoma, with a median duration of response of approximately 1 year1-3. Immunotherapy with antibodies to programmed death 1 (PD-1) produces lower response rates but with long response duration. Preclinical models suggest that combining BRAF and MEK inhibitors with PD-1 blockade therapy improves antitumor activity4-6, which may provide additional treatment options for patients unlikely to have long-lasting responses to either mode of therapy alone. We enrolled 15 patients with BRAFV600-mutated metastatic melanoma in a first-in-human clinical trial of dabrafenib, trametinib and pembrolizumab ( NCT02130466 ). Eleven patients (73%) experienced grade 3/4 treatment-related adverse events, the most common being elevation of liver function tests and pyrexia, most of which resolved with drug interruption or discontinuation of either the anti-PD-1 antibody or the targeted therapy combination. Eleven patients (73%; 95% confidence interval = 45-92%) had an objective response, and six (40%; 95% confidence interval = 16-68%) continued with a response at a median follow-up of 27 months (range = 10.3-38.4+ months) for all patients. This study suggests that this triple-combined therapy may benefit a subset of patients with BRAFV600-mutated metastatic melanoma by increasing the frequency of long-lasting antitumor responses.

Interleukin 32 expression in human melanoma.

BACKGROUND: Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma. METHODS: We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5' upstream region of the human IL32 gene into a luciferase reporter vector. RESULTS: A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, ß, γ) of IL32. Cis-acting elements within this 5' upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature. CONCLUSIONS: Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5' upstream region.

A Pilot Trial of the Combination of Transgenic NY-ESO-1-reactive Adoptive Cellular Therapy with Dendritic Cell Vaccination with or without Ipilimumab.

PURPOSE: Transgenic adoptive cell therapy (ACT) targeting the tumor antigen NY-ESO-1 can be effective for the treatment of sarcoma and melanoma. Preclinical models have shown that this therapy can be improved with the addition of dendritic cell (DC) vaccination and immune checkpoint blockade. We studied the safety, feasibility, and antitumor efficacy of transgenic ACT with DC vaccination, with and without CTLA-4 blockade with ipilimumab. PATIENTS AND METHODS: Freshly prepared autologous NY-ESO-1-specific T-cell receptor (TCR) transgenic lymphocytes were adoptively transferred together with NY-ESO-1 peptide-pulsed DC vaccination in HLA-A2.1-positive subjects alone (ESO, NCT02070406) or with ipilimumab (INY, NCT01697527) in patients with advanced sarcoma or melanoma. RESULTS: Six patients were enrolled in the ESO cohort, and four were enrolled in the INY cohort. Four out of six patients treated per ESO (66%), and two out of four patients treated per INY (50%) displayed evidence of tumor regression. Peripheral blood reconstitution with NY-ESO-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Tracking of transgenic T cells to the tumor sites was demonstrated in on-treatment biopsies via TCR sequencing. Multiparametric mass cytometry of transgenic cells demonstrated shifting of transgenic cells from memory phenotypes to more terminally differentiated effector phenotypes over time. CONCLUSIONS: ACT of fresh NY-ESO-1 transgenic T cells prepared via a short ex vivo protocol and given with DC vaccination, with or without ipilimumab, is feasible and results in transient antitumor activity, with no apparent clinical benefit of the addition of ipilimumab. Improvements are needed to maintain tumor responses.

Multi-stage Differentiation Defines Melanoma Subtypes with Differential Vulnerability to Drug-Induced Iron-Dependent Oxidative Stress.

Malignant transformation can result in melanoma cells that resemble different stages of their embryonic development. Our gene expression analysis of human melanoma cell lines and patient tumors revealed that melanoma follows a two-dimensional differentiation trajectory that can be subclassified into four progressive subtypes. This differentiation model is associated with subtype-specific sensitivity to iron-dependent oxidative stress and cell death known as ferroptosis. Receptor tyrosine kinase-mediated resistance to mitogen-activated protein kinase targeted therapies and activation of the inflammatory signaling associated with immune therapy involves transitions along this differentiation trajectory, which lead to increased sensitivity to ferroptosis. Therefore, ferroptosis-inducing drugs present an orthogonal therapeutic approach to target the differentiation plasticity of melanoma cells to increase the efficacy of targeted and immune therapies.

Genetic Mechanisms of Immune Evasion in Colorectal Cancer.

To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability-high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of B2M and HLA genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/ß-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/ß-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion.Significance: This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. Cancer Discov; 8(6); 730-49. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Integrated molecular analysis of Tamoxifen-resistant invasive lobular breast cancer cells identifies MAPK and GRM/mGluR signaling as therapeutic vulnerabilities.

Invasive lobular breast cancer (ILC) is an understudied malignancy with distinct clinical, pathological, and molecular features that distinguish it from the more common invasive ductal carcinoma (IDC). Mounting evidence suggests that estrogen receptor-alpha positive (ER+) ILC has a poor response to Tamoxifen (TAM), but the mechanistic drivers of this are undefined. In the current work, we comprehensively characterize the SUM44/LCCTam ILC cell model system through integrated analysis of gene expression, copy number, and mutation, with the goal of identifying actionable alterations relevant to clinical ILC that can be co-targeted along with ER to improve treatment outcomes. We show that TAM has several distinct effects on the transcriptome of LCCTam cells, that this resistant cell model has acquired copy number alterations and mutations that impinge on MAPK and metabotropic glutamate receptor (GRM/mGluR) signaling networks, and that pharmacological inhibition of either improves or restores the growth-inhibitory actions of endocrine therapy.