Angel Garcia-Diaz

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.).

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.

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

SUMMARY 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.

Response to Programmed Cell Death-1 Blockade in a Murine Melanoma Syngeneic Model Requires Costimulation, CD4, and CD8 T Cells.

Although blockade of the PD-1 pathway has been successfully used to treat various cancers, how this modulates host–tumor interactions is not well understood. Additional mechanisms beyond licensing the final effector phase of killer T cells were identified. The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues, and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor–host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN−/−). Anti–PD-1 or anti–PD-L1 therapy engendered strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. PD-L1 expression did not differ between the three models at baseline or upon interferon stimulation. Whereas mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T cells, as well as CD28 and CD80/86 costimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor-associated macrophages in the tumors after PD-1 blockade. Compared with YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in expression of chemokine-trafficking genes that are related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells and costimulation that is mediated by dendritic cells and macrophages. Cancer Immunol Res; 4(10); 845–57. ©2016 AACR.

Single-cell analysis resolves the cell state transition and signaling dynamics associated with melanoma drug-induced resistance

Significance This work provides biophysical insights into how BRAF mutant melanoma cells adapt to the stress of MAPK inhibition via a series of reversible phenotypic transitions toward drug-tolerant or drug-resistant cell states enriched for neural-crest factors and mesenchymal signatures. This adaptation is influenced by cell phenotype-specific drug selection and cell state interconversion, but not selection of genetically resistant clones. A panel of functional proteins, analyzed at the single-cell level, pointed to signaling network hubs that drive the initiation of the melanoma cell adaptive transition. Targeting those hubs halted the transition and arrested resistance development. Continuous BRAF inhibition of BRAF mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived BRAFV600-mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.

Innate resistance of PD-1 blockade through loss of function mutations in JAK resulting in inability to express PD-L1 upon interferon exposure

PD-L1-negative tumors assessed by immunohistochemistry often still respond to PD-1 blockade. PD-L1 is inducible by interferon, therefore, absolute negative tumors are the ones unable to up-regulate PD-L1 in response to interferons. Genetic mutations in the interferon receptor signaling pathway leading to loss of PD-L1 up-regulation were hypothesized to exhibit innate resistance to PD-1 blockade.

Isolation and characterization of NY-ESO-1–specific T cell receptors restricted on various MHC molecules

Significance T immune cells can be engineered to express tumor-specific T cell receptor (TCR) genes and thereby kill cancer cells. This approach—termed TCR gene therapy—is effective but can cause serious adverse events if the target is also expressed in healthy, noncancerous tissue. NY-ESO-1 is a tumor-specific antigen that has been targeted successfully and safely through TCR gene therapies for melanoma, synovial sarcoma, and myeloma. However, trials to date have focused exclusively on a single NY-ESO-1–derived epitope presented on HLA-A*02:01, limiting application to patients expressing that allele. In this work, we isolate TCRs that collectively recognize multiple NY-ESO-1–derived epitopes presented by multiple MHC alleles. We thereby outline a general approach for expanding targeted immunotherapies to more diverse MHC haplotypes. Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2–restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.

Abstract 3765: IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR

T cell receptor (TCR) engineered adoptive T cell transfer (ACT) has shown remarkable antitumor efficacy in several clinical trials. However, low persistence of modified cells limits long-term clinical responses. To overcome this hurdle, we propose a clinical trial co-administering genetically modified T cells and stem cells both expressing an NY-ESO-1 TCR such that the engrafted stem cells generate a source for constant renewal of modified T cells. Here we report a pre-clinical IND-enabling study performed at UCLA under Good Laboratory Practice (GLP) compliance to assess whether co-administration impacts (I) safety; (II) engraftment and cell lineage differentiation of gene modified stem cells; and (III) persistence of adoptively transferred T cells and stem cell-derived progeny. We performed 12 optimization studies to define the optimal conditions for TCR gene modified ACT and TCR gene modified hematopoietic stem cell (HSC) bone marrow transplantation (BMT). Sixty-four HLA-A2/kb transgenic mice were myelodepleted and received syngeneic BMT with Lineage depleted bone marrow (Lin-) cells transduced with the LV-NYESO-1 TCR/sr39TK and ACT with T cells transduced with the RV-NYESO-1 TCR. Control groups were as follows: untreated mice, mice receiving mock transduced Lin- cells and T cells, mice receiving transduced Lin- cells and mock transduced T cells, and mice receiving mock transduced Lin- cells and transduced T cells (n = 16 per group). Overall survival at 3 months was 87.5%; no significant differences in survival were observed among cohorts. After BMT we observed a decrease in body weight, elevation in creatinine kinase and transaminases, and gonadal germ cell ablation in all cohorts. Three months after BMT, all blood cell lineages were reconstituted in surviving mice. Using digital droplet PCR and flow cytometry, we confirmed that transduced stem cells engrafted and their progeny persisted long term. In the bone marrow, NY-ESO-1 TCR was expressed intracellularly among progenitor cells (Lin-, LSK and HSC) as well as all hematopoietic cell lineages within the spleen (CD8+ T cells, CD4+ T cells, NKT cells, B cells and granulocytes). Co-administration with gene modified T cells and stem cells did not affect engraftment, cell lineage differentiation or persistence of the gene modified stem cells. Moreover, co-administration with stem cells did not affect persistence of adoptively transferred T cells. These data demonstrate that 1) NY-ESO-1 TCR genetically modified stem cells engraft and differentiate into all hematopoietic cell lineage progeny, which persists at 3 months; 2) adoptively transferred NY-ESO-1 TCR T cells persist at 3 months; 3) co-administration of stem cells and T cells genetically modified to express an NY-ESO-1 TCR is safe and does not negatively impact stem cell engraftment, lineage differentiation and progeny persistence or T cell persistence. Citation Format: Cristina Puig-Saus, Giulia Parisi, Paige Krystofinski, Angel Garcia-Diaz, Salemiz Sandoval, James McCabe, Ruixue Zhang, Gardenia Cheung-Lau, Nhat Truong, Justin Saco, Sara Komenan, Agustin Vega-Crespo, Mignonette H Macabali, Begona Comin-Anduix, Beata Berent-Maoz, Donald Kohn, Paula Kaplan-Lefko, Antoni Ribas. IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR [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 3765. doi:10.1158/1538-7445.AM2017-3765

Abstract 5013: Toward better selection of PD-1 blockade responders through direct markers of adaptive immune resistance

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction. The role of PD-L1 as a predictive biomarker for response to PD-1 blockade is unclear. Emerging evidence, including our findings reported here, suggests that PD-L1 expression is important only when induced by the presence of T cells producing interferons, in what is termed adaptive immune resistance (Tumeh, Nature 2014). Therefore, studying PD-L1 expression in response or not to interferons may define molecular mechanisms to select patients whose tumors are incapable to respond to T cell infiltration by upregulating PD-L1. Experimental procedures. After the optimization process, we exposed 50 established melanoma cell lines to interferons (alpha, beta and gamma) and measured PD-L1, pSTAT1 (Y701), pSTAT3, pSTAT5, pSTAT6, NF-kB, pERK (1/2), pAKT, pP38, cleaved PARP, CyD3 expression level by flow cytometry (FACS LSRII) and PD-L1 as well as STAT1 transcript level was checked by quantitative real time PCR. Western blots were performed to further analyze interferon receptor signaling in selected cells lines. Results. Of 50 cell lines tested, 44 responded to at least one interferon ranging from 2 to 10-fold increase in PD-L1 compared to baseline expression, and 5 cell lines failed to respond to interferons with PD-L1 upregulation. Measurement of PD-L1 transcript level by qPCR as well as surface expression level by flow cytometry demonstrated that PD-L1 transcript level was up-regulated close to 20 fold by IFNγ in 30 minutes for two representatives of good PD-L1 responders. Phosphoflow experiments confirmed that 3 representative good responding cell lines had significant up-regulation of pSTAT1 compared to the baseline. However, among non-PD-L1 responding cell lines M412B showed significant change in pSTAT1, but WM1366 and M395 did not both, by phosphoflow and western blot analyses. M368 is a cell line that did not up-regulate PD-L1 upon exposure to interferon gamma, but it did up-regulate PD-L1 when exposed to type I interferons (alpha and beta). This cell line showed loss of JAK2 expression. WM1366 showed low JAK2 expression with loss of pJAK2 and SOCS1 overexpression, which is consistent with non-significant up-regulation of PD-L1 upon interferon treatment. Conclusions. PD-L1 is inducible in the majority of melanoma cell lines tested, but approximately 10% of them are not able to up-regulate PD-L1 expression upon interferon exposure. JAK-STAT pathway is critically important in regulation of inducible expression of PD-L1, and we have defined molecular defects in individual cell lines, which did not up-regulate PD-L1 expression. This information may be important to assess clinical data from PD-1/L1 blockade trials as to whether intact interferon receptor pathway inducible response may better predict the response of anti-PD-1/L1 immunotherapy than baseline PD-L1 levels. Citation Format: Daniel S. Shin, Angel Garcia-Diaz, Helena Escuin-Ordinas, Nicolaos J. Palaskas, Sara M. Komenan, Thomas G. Graeber, Begonya Comin-Anduix, Antoni Ribas. Toward better selection of PD-1 blockade responders through direct markers of adaptive immune resistance. [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 5013. doi:10.1158/1538-7445.AM2015-5013