Antitumor Immune Responses in B2M-Deficient Cancers.

ß2-microglobulin (B2M) is a critical component of the MHC class I molecule and is required to present tumor antigens to T cells. Its loss results in acquired resistance to immune checkpoint blockade (ICB) therapies. However, there have been well-documented cases of B2M-inactivated tumors responding to ICB, justifying investigation of how an antitumor immune response can be generated to tumors without surface MHC class I. We knocked out B2M in three murine models with varying baseline MHC class I expression and sensitivity to anti-programmed death receptor (PD-1) therapy and analyzed the immune responses. MC38 and YUMMER2.1 without B2M responded to anti-PD-1 alone or with an IL2 agonist, and this was mediated by CD4+ T cells and natural killer (NK) cells. The more aggressive B16 without B2M expression only partially responded to the IL2 agonist, and this was dependent on NK cells. When analyzing nearly 300 pretreatment biopsies from patients with melanoma receiving PD-1 blockade-based therapies, we found infrequent B2M mutations or homozygous loss but more frequent LOH or copy-number gains. B2M LOH was enriched in biopsies from patients without response to therapy, and these biopsies were more frequently infiltrated by activated NK cells. We conclude that in the absence of B2M, activation of CD4+ T cells and NK cells can mediate responses to murine models of PD-1 blockade therapy. In addition, in human melanoma, the intratumoral presence of activated NK cells upon partial B2M loss likely selects against tumor escape through low surface MHC class I expression.

Remodeling of the tumor microenvironment through PAK4 inhibition sensitizes tumors to immune checkpoint blockade.

PAK4 inhibition can sensitize tumors to immune checkpoint blockade (ICB) therapy, however, the underlying mechanisms remain unclear. We report that PAK4 inhibition reverses immune cell exclusion by increasing the infiltration of CD8 T cells and CD103+ dendritic cells (DCs), a specific type of DCs that excel at cross-presenting tumor antigens and constitute a source of CXCL10. Interestingly, in melanoma clinical datasets, PAK4 expression levels negatively correlate with the presence of CCL21, the ligand for CCR7 expressed in CD103+ DCs. Furthermore, we extensively characterized the transcriptome of PAK4 knock out (KO) tumors, in vitro and in vivo, and established the importance of PAK4 expression in the regulation of the extracellular matrix, which can facilitate immune cell infiltration. Comparison between PAK4 wild type (WT) and KO anti-PD-1 treated tumors revealed how PAK4 deletion sensitizes tumors to ICB from a transcriptomic perspective. In addition, we validated genetically and pharmacologically that inhibition of PAK4 kinase activity is sufficient to improve anti-tumor efficacy of anti-PD-1 blockade in multiple melanoma mouse models. Therefore, this study provides novel insights into the mechanism of action of PAK4 inhibition and provides the foundation for a new treatment strategy that aims to overcome resistance to PD-1 blockade by combining anti-PD-1 with a small molecule PAK4 kinase inhibitor.

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.

Uncoupling interferon signaling and antigen presentation to overcome immunotherapy resistance due to JAK1 loss in melanoma.

Defects in tumor-intrinsic interferon (IFN) signaling result in failure of immune checkpoint blockade (ICB) against cancer, but these tumors may still maintain sensitivity to T cell-based adoptive cell therapy (ACT). We generated models of IFN signaling defects in B16 murine melanoma observed in patients with acquired resistance to ICB. Tumors lacking Jak1 or Jak2 did not respond to ICB, whereas ACT was effective against Jak2 KO tumors, but not Jak1 KO tumors, where both type I and II tumor IFN signaling were defective. This was a direct result of low baseline class I major histocompatibility complex (MHC I) expression in B16 and the dependency of MHC I expression on either type I or type II IFN signaling. We used genetic and pharmacologic approaches to uncouple this dependency and restore MHC I expression. Through independent mechanisms, overexpression of NLRC5 (nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 5) and intratumoral delivery of BO-112, a potent nanoplexed version of polyinosinic:polycytidylic acid (poly I:C), each restored the efficacy of ACT against B16-Jak1 KO tumors. BO-112 activated double-stranded RNA (dsRNA) sensing (via protein kinase R and Toll-like receptor 3) and induced MHC I expression via nuclear factor κB, independent of both IFN signaling and NLRC5. In summary, we demonstrated that in the absence of tumor IFN signaling, MHC I expression is essential and sufficient for the efficacy of ACT. For tumors lacking MHC I expression due to deficient IFN signaling, activation of dsRNA sensors by BO-112 affords an alternative approach to restore the efficacy of ACT.

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.

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.

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.

Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression.

PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.

Primary Resistance to PD-1 Blockade Mediated by JAK1/2 Mutations.

Loss-of-function mutations in JAK1/2 can lead to acquired resistance to anti-programmed death protein 1 (PD-1) therapy. We reasoned that they may also be involved in primary resistance to anti-PD-1 therapy. JAK1/2-inactivating mutations were noted in tumor biopsies of 1 of 23 patients with melanoma and in 1 of 16 patients with mismatch repair-deficient colon cancer treated with PD-1 blockade. Both cases had a high mutational load but did not respond to anti-PD-1 therapy. Two out of 48 human melanoma cell lines had JAK1/2 mutations, which led to a lack of PD-L1 expression upon interferon gamma exposure mediated by an inability to signal through the interferon gamma receptor pathway. JAK1/2 loss-of-function alterations in The Cancer Genome Atlas confer adverse outcomes in patients. We propose that JAK1/2 loss-of-function mutations are a genetic mechanism of lack of reactive PD-L1 expression and response to interferon gamma, leading to primary resistance to PD-1 blockade therapy. SIGNIFICANCE: A key functional result from somatic JAK1/2 mutations in a cancer cell is the inability to respond to interferon gamma by expressing PD-L1 and many other interferon-stimulated genes. These mutations result in a genetic mechanism for the absence of reactive PD-L1 expression, and patients harboring such tumors would be unlikely to respond to PD-1 blockade therapy. Cancer Discov; 7(2); 188-201. ©2016 AACR.See related commentary by Marabelle et al., p. 128This article is highlighted in the In This Issue feature, p. 115.

Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma.

BACKGROUND: Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.).

Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma.

Cell-free circulating tumour DNA (ctDNA) in plasma has been shown to be informative of the genomic alterations present in tumours and has been used to monitor tumour progression and response to treatments. However, patients with brain tumours do not present with or present with low amounts of ctDNA in plasma precluding the genomic characterization of brain cancer through plasma ctDNA. Here we show that ctDNA derived from central nervous system tumours is more abundantly present in the cerebrospinal fluid (CSF) than in plasma. Massively parallel sequencing of CSF ctDNA more comprehensively characterizes the genomic alterations of brain tumours than plasma, allowing the identification of actionable brain tumour somatic mutations. We show that CSF ctDNA levels longitudinally fluctuate in time and follow the changes in brain tumour burden providing biomarkers to monitor brain malignancies. Moreover, CSF ctDNA is shown to facilitate and complement the diagnosis of leptomeningeal carcinomatosis.

Bevacizumab in advanced breast cancer: a new model for the assessment of activity in non-first-line treatment regimens.

The activity of bevacizumab (BVZ) in advanced lines is not well known. In the treatment of metastatic breast cancer, the response rate and time to treatment failure (TTF) decrease with progression through successive therapeutic lines. The objective of this study was to compare BVZ activity in advanced treatment lines with that achieved in the previous line in routine clinical practice. Ninety-six patients who had received BVZ treatment in second or subsequent treatment lines were selected from five Spanish hospitals. Analysis was carried out of the differences in TTF and response rate in the lines with BVZ and those in earlier lines. Data analysis was carried out in two different ways: (a) by comparing treatment groups according to the treatment line received, using a Cox regression model with random effects, and the McNemar test to analysis the response rate, and (b) by comparing intrapatient data, using the Wilcoxon signed-rank test. In 62 patients, the TTF (adjusted for treatment line) was longer in the BVZ treatment line than that in the previous line. In the BVZ lines, there was a significant reduction in the probability of treatment failure [hazard ratio 0.52; 95% confidence interval (CI) 0.38-0.71]. The median TTF was 4.27 months (95% CI 3.7-5) in the previous line and 6.18 months (95% CI 5.5-7.93) in the BVZ line. The percentage of patients with an objective response was 33.3% in the previous lines and 52.1% (P=0.005) in the BVZ line. Contrary to expectation, more patients showed better results with the BVZ line than with the previous line. BVZ treatment in advanced lines improves the results obtained in previous treatment lines. This suggests that BVZ is active in advanced lines and that it produces favourable changes in the natural history of patients with metastatic breast carcinoma.

Implication of breast cancer phenotype for patients with leptomeningeal carcinomatosis.

BACKGROUND: We aimed to study the implications of breast cancer (BC) subtypes for the development and prognosis of leptomeningeal carcinomatosis (LC). PATIENTS AND METHODS: Data from the breast cancer patients diagnosed with LC between 2005 and 2010 were retrieved. Patients were classified in luminal A, B, HER2 positive and triple negative (TN) and their BC diagnosis, treatment, and outcome were analyzed according to each subtype. Pearson's chi-square and Fisher's exact test were used for categorical variables. Survival analyses were performed by Kaplan-Meier method and compared with the log-rank test. RESULTS: A total of 38 BC patients were identified, with a median age of 54.8 years (range 36-79). The proportion of luminal A, B, HER2 positive and TN was 18.4%, 31.6%, 26.3% and 23.7%, respectively. LC was the first evidence of metastatic disease in 5 BC patients. Twenty patients received the systemic chemotherapy, with 16 (80%) whole brain radiotherapy (WBRT). Nine patients received only WBRT. TN patients had the shorter interval between metastatic breast cancer diagnosis and the development of LC. Median survival after the diagnosis of LC (OSLC) was 2.6 months (range 1.2-6.4), and did not differ across breast cancer subtypes. In univariate analysis, performance status (ECOG = 0-2) and chemotherapy were prognostic for OSLC, but only the treatment stood as an independent prognostic factor in multivariate analysis. CONCLUSIONS: Breast cancer subtype influences the timing of LC appearance, but not OSLC. Patients with LC from breast cancer should be offered systemic treatment, as it appears to associate with the improved outcome. New therapeutic strategy, including, targeted and intrathecal therapy are deserved for BC patients with LC.

Serum tumor markers CEA, CYFRA21-1, and CA-125 are associated with worse prognosis in advanced non-small-cell lung cancer (NSCLC).

BACKGROUND: Serum tumor markers are considered a negative prognostic factor in early-stages NSCLC but its role in advanced disease is controversial. The aim of this study is to analyze the prognostic value of tumor markers in advanced NSCLC. PATIENTS AND METHODS: Two hundred and seventy seven patients diagnosed in our institution were retrospectively reviewed. Baseline prognostic factors analyzed were gender, histology and brain metastases. RESULTS: Baseline patients characteristics: median age 63 years (30-81 years); males 84.4%, stage IV: 61.7%; adenocarcinoma 38.6%, squamous carcinoma 22.4%. High levels of CEA, CYFRA21-1, and CA125 levels were detected in 179 (55.9%), 119 (65%), and 129 (46.6%) patients respectively. Significant higher levels of CEA and CA125 at baseline were present in adenocarcinoma (P < .05). PFS in patients with elevated CEA, CYFRA21-1, and CA125 was 5.3 months (m), 3.5 m and 4.6 m versus 7.4 m, 6.2 m and 7.5 m in patients with normal levels (P < .05). The OS in patients with high and normal levels of tumor markers was 10.0 m vs 14.0 m (P = 0.085) for CEA; 5.6 vs 12.1 m for CYFRA21-1 (P = .002), and 8.7 vs 14.0 (P = .03) for CA125. In the multivariate analysis high levels of tumor markers, histology and clinical stage were significant correlated with worse prognostic. Patients with all the tumor markers elevated presented the worst prognosis (3.6 m for PFS and 7.1 m for OS, P < .001). CONCLUSION: In our analysis, high levels of tumor markers at baseline are correlated with worse survival in stage III-IV NSCLC patients.