Metastatic

Anti–PD-1 therapy has transformed the management of NSCLC, but only a subset of patients respond to anti–PD-1 therapy, and responses are rarely curative. Understanding the effects of anti–PD-1 therapy on the composition and functional state of tumor-associated immune subsets can identify mechanisms of response and resistance to anti–PD-1 therapy and provide insights into rational combination strategies to improve upon response rates. Although the effects of anti–PD-1 therapy within the tumor immune microenvironment in preclinical and human subjects have been previously reported, we propose to broadly characterize the effects of anti–PD-1 therapy on the tumor draining lymph nodes (TDLN). Studying the TDLN is particularly valuable because it is the primary sites at which the tumor antigen exposure and the main site of immune T-cell differentiation. To determine the effect of ICI response in the lymph node, we have created an immunocompetent murine model that recapitulates early-stage treatment and outcomes in patients. Additionally, we have characterized TDLN T cells from early-stage cancer patients and have been able to recapitulate our preclinical findings of unique memory T-cell subsets. Our preliminary results of the immune cell type composition, checkpoint expression, and cytokine expression within TDLNs suggest that there are major remodeling events within the TDLN mediated by anti–PD-1 therapy that are critical for conferring effective antitumor long-term immune responses. We demonstrate that unique memory T cell subsets undergo significant activation, expansion, and maturation in response to anti–PD-1 therapy, to shape antitumor programming in TDLNs. Importantly, our proposed study will provide a methodological demonstration in both a murine model and human TDLN that simultaneous uses Flowcytometry, functional immune assays and single cell sequencing that will enable deep interrogation of the immune profile to PD-1 inhibition resistant and responsive patients.

Our laboratory has demonstrated the potency of the abscopal effect in studies showing that high-dose radiation therapy delivered to primary tumors in combination with dual immune checkpoint blockade can significantly inhibit the growth of satellite tumors. The implication of such results is that radiation therapy has remote effects which are immune-mediated and that new radiation protocols can be devised to exploit our naturally occurring defenses against cancer. Previous studies have shown that immune-mediated tumoricidal effects can be augmented by combining high-dose radiation therapy delivered to a primary tumor site with low-dose radiotherapy (LDRT) targeting secondary tumors. Tumor response is further enhanced if systemic agents are employed to induce immune-checkpoint blockade (ICB). Such protocols employing conventional LDRT are known to reduce the number of regulatory T cells (Tregs) and to activate natural killer cells, thereby modulating the tumor microenvironment (TME). Our laboratory project seeks to test a modification of LDRT called pulsed low-dose-rate radiotherapy (PLDR). By providing radiation in repeated very low doses, the PLDR regimen may reduce the encasement of tumors with stromal elements, thus removing a barrier to infiltrating immune-reactive cells. We propose to use the immunocompetent lung cancer murine model which we developed for our earlier research combining HDRT with anti-PD1 and anti-CTLA-4 antibodies. HDRT will again be delivered to primary tumors in combination with dual ICB. Additionally, LDRT will be delivered to secondary tumors, both in conventional form and as PLDR. The results will thus test an innovative method for exploiting the emerging synergies of radiation and ICB. A demonstration of the superior therapeutic utility of PLDR in our animal model would be of immediate relevance to clinical trials seeking improved strategies for treating patients with metastatic lung cancer.

Despite recent advances, locally advanced non-small cell lung cancer (LA-NSCLC) continues to have a high degree of morbidity and mortality related to both disease progression and sequelae from treatment. The purpose of this study is to develop and evaluate a more personalized radiation therapy (RT) approach with the potential to reduce time and financial burden to patients and increase tumor control. Patients undergoing fractionated RT with concurrent systemic therapy for LA-NSCLC will be treated to a total dose of 60 Gy with the number of fractions determined by ability to meet key dose constraints resulting in a shorter treatment course and higher biologic effective dose (BED) among those treated with shorter fractionation. To reduce the iterative time to determine fractionation schedule and standardize the"‘isotoxic hypofractionation" approach, we will employ a previously developed automated RT planning technique after delineation of the tumor and organs at risk. Fifty patients will be enrolled in a prospective non-randomized 2-stage trial to test the primary objective of whether isotoxic hypofractionation results in an acceptable rate of grade 2 or higher toxicity. Secondary endpoints will include G3+ toxicity, local control, progression free survival and overall survival. Finally, to better inform future "isotoxic" dosing strategies we will evaluate additional predictors of radiation toxicity using established retrospective cohorts through the Stanford Cancer Institute and the Veteran’s Affairs Information and Computing Infrastructure (VINCI).

The targeted-therapy (TT) osimertinib is frontline standard-of-care for patients with advanced non-small cell lung cancer (NSCLC) harboring mutations in the epidermal growth factor receptor (EGFR) gene. However, nearly all patients develop resistance, at which point treatments are limited. While revolutionary for many cancers, anti-PD-(L)1 immune checkpoint blockade (ICB) has been ineffective in EGFR-mutated(m) NSCLC. Targeting angiogenesis, and specifically vascular endothelial growth factor (VEGF), may be key to changing this. VEGF promotes immunosuppression through multiple mechanisms including inhibition of cytotoxic T-cell trafficking and promotion of regulatory T-cell proliferation. Pre-clinical and clinical evidence has revealed activated EGFR to be a dominant regulator of angiogenesis in EGFRm NSCLC, with increased expression of VEGF and hypoxia inducible factor-1α observed in EGFRm compared to wild-type NSCLC. Clinically, combination chemotherapy with anti-PD-L1 and VEGF blockade has shown efficacy signals in EGFRm NSCLC. However, whether upregulated VEGF potentiates the uninflamed tumor immune microenvironment (TIME), and underlies the observed synergy of these therapies in EGFRm NSCLC, remains unexplored. It is further unknown if VEGF inhibition can be combined with PD-1 blockade to promote benefit in the absence of chemotherapy, or whether blockade of additional checkpoints can further expand anti-tumor immune populations, specifically NK cells, which appear to be upregulated in EGFRm NSCLC responsive to ICB. To address these impactful questions, we have designed a single-arm translationally-focused phase 2 clinical trial assessing the efficacy of tiragolumab (anti-TIGIT) plus atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF) in advanced TT-refractory, ICB-naïve, EGFRm NSCLC. Critically, the proposed analyses detailed herein of tumor and blood samples obtained pre- and on-treatment will focus on (1) immunopathologic features of ICB resistance in EGFRm-NSCLC and (2) TIME infiltration and immunologic reprogramming imparted by these therapies. The findings from this study will help to define the innate barriers preventing induction of durable anti-tumor immunity by ICB in EGFRm NSCLC, and identify strategies to overcome them.

Rearrangement of the BRAF gene results in a potent oncogene that is a driver in 2.8% of NSCLC with BRAF alterations. To date there is no approve targeted therapy for any cancer type with BRAF fusions. Studies by our group and others have shown that mutant-specific BRAF kinase inhibitors do no inhibit BRAF fusion kinases. Instead, pan-RAF inhibitors seem more effective as therapy for cancer cells harboring these fusions. We therefore hypothesize that targeting BRAF fusions with pan-RAF inhibitors are likely to be effective therapy for this molecularly defined subset of NSCLC. Our goal in this proposal is to generate the preclinical data necessary to propel a phase 1 clinical trial of a pan-RAF inhibitor specific to patients with NSCLC harboring a BRAF fusion. We have generated seven preclinical disease models with BRAF rearrangement, including two NSLC patient-derived xenograft models with matching cell lines that will allow this project to go forward immediately. In Aim 1 we propose to generate additional NSCLC PDX, organoid and cell line models that will expand the platforms available to examine the efficacy of these BRAF inhibitors. We will test the efficacy of three pan-RAF inhibitors (LY3009120, belvarafenib, KIN-2787) in vitro and in vivo in Aim 2. We believe that combination therapy may eventually be necessary to extend the duration of response to BRAF targeted therapy. To find candidates that can be exploited for combination therapy, in Aim 3 we will map the signaling pathways that are activated by BRAF fusions in isogenic cell lines and inhibited specifically by BRAF inhibitors in cells with BRAF fusions. Given that resistance to therapy is inevitable, we will seek to generate resistance to the three pan-RAF inhibitors in vitro and in vivo and then identify and validate potential mechanisms of resistance. To be able to bring the goals of this proposal to fruition, I have assembled a team with clinical, translational and basic research expertise to help guide me. As a thoracic oncologist with clinical trial expertise, I am ideally situated to translate the findings of this study to the clinic.

Genomic discoveries of driver-gene alterations in lung cancer have led to development of effective target therapeutics; however patients who initially respond to the therapy inevitably experience regrowth of the disease. The reversible drug-tolerant persister (DTP) stage, where cells enter a quiescence, is gaining attention as a major source of non-genetic drug resistance. In our EGFR-TKI-induced DTP model, we found a marked overload of repressive chromatin modification (H3K9me3 and H3K27me3) reminiscent of a quiescent state. In Aim1, we seek to characterize the epigenomic dynamics of DTP stage in RET-positive lung cancer under Selpercatinib treatment, and determine the roles of histone-modifying enzymes on maintaining DTP stage as potential combinatorial therapeutic targets. The process of DTP development requires terminally differentiated cells to regain plasticity and undergo re-differentiation, while the origin of DTP plasticity remains unknown. A recent scRNA-seq study from lung cancer biopsies representing DTP states showed an elevated alveolar signature. During lung regeneration, an alveolar lineage factor HOPX is required for the AT1 plasticity to regenerate AT2 cells upon injury, suggesting a differentiation potential of HOPX(+) cells. Our data showed HOPX is an early marker for treatment-induced DTP and necessary for DTP development, where our scATAC-seq data exhibited increased epigenomic heterogeneity. We hypothesize DTP contains heterogeneous subpopulations that hijack developmental pathways to sustain plasticity for subsequent differentiation. In Aim2, we will determine the role of HOPX and associated developmental pathways in RET-induced DTP model, to identify novel targets determining the DTP plasticity thereby preventing relapse from heterogeneous pathways to acquire Selpercatinib resistance.

Fusions in the RET proto-oncogene account for ~2% of non-small cell lung cancers (NSCLC). Despite initial failures tied to the use of multi-kinase inhibitors, recent selective RET inhibitors have proven remarkably more successful. However, patients eventually recur on these therapies, highlighting the need for other therapeutic alternatives. Immunotherapies have been unsuccessful in patients harboring RET fusions, likely in part due to their low tumor mutational burden. Neoantigens are immunogenic antigens which are derived from somatic mutations, resulting in antigen-specific killing by T lymphocytes. Importantly, insertions, deletions, and gene fusions may exhibit increased immunogenicity, due to their accrued dissimilarity with unmutated transcripts and proteins, which may provide a unique opportunity to target RET fusions using T cell receptor engineering approaches. Accordingly, we hypothesize that RET fusions are immunogenic and can be effectively recognized using T cell receptor engineering approaches. To confirm this hypothesis, we propose to identify RET fusion-derived neoantigens, identify and isolate the T cells which recognize them, and engineer T cells to kill targets in vitro. Through this work, we aim to generate a library of TCRs which could be used off-the-shelf to target the two dominant RET fusions (KIF5B-RET=75%; CCDC6-RET=25%) and 10 most prevalent HLA alleles in the United States, in order to afford new therapeutic alternatives to the overwhelming majority of patients harboring RET fusions in NSCLC.