Autochthonous Tumors Research Articles

Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

BackgroundThe prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. It has been suggested that the adenosine pathway contributes to the ability of PDAC to evade the immune...

Reprogramming the immunosuppressive tumor microenvironment results in successful clearance of tumors resistant to radiation therapy and anti-PD-1/PD-L1

ABSTRACT Despite breakthroughs in immune checkpoint inhibitors (ICI), the majority of tumors, including those poorly infiltrated by CD8+ T cells or heavily infiltrated by immunosuppressive immune effector cells, are unlikely to result in clinically meaningful tumor responses. Radiation therapy (RT) has been combined with ICI to potentially overcome this resistance and improve response rates but reported clinical trial results have thus far been disappointing. Novel approaches are required to overcome this resistance and reprogram the immunosuppressive tumor microenvironment (TME) and address this major unmet clinical need. Using diverse preclinical tumor models of prostate and bladder cancer, including an autochthonous prostate tumor (Pten−/−/trp53−/−) that respond poorly to radiation therapy (RT) and anti-PD-L1 combinations, the key drivers of this resistance within the TME were profiled and used to develop rationalized combination therapies that simultaneously enhance activation of anti-cancer T cell responses and reprogram the immunosuppressive TME. The addition of anti-CD40mAb to RT resulted in an increase in IFN-y signaling, activation of Th−1 pathways with an increased infiltration of CD8+ T-cells and regulatory T-cells with associated activation of the CTLA−4 signaling pathway in the TME. Anti-CTLA−4mAb in combination with RT further reprogrammed the immunosuppressive TME, resulting in durable, long-term tumor control. Our data provide novel insights into the underlying mechanisms of the immunosuppressive TME that result in resistance to RT and anti-PD−1 inhibitors and inform therapeutic approaches to reprogramming the immune contexture in the TME to potentially improve tumor responses and clinical outcomes.

EPEN-01. ZFTA-RELA FUSION PROGRAMS CONVERGE ON YAP PATHWAY ACTIVATION IN EPENDYMOMA

Abstract Approximately two thirds of supratentorial ependymoma are driven by the ZFTA-RELA gene fusion. ZFTA is a recently described gene with a role in DNA binding and transcriptional regulation, while RELA (p65) is a known principal mediator of the canonical NFkB pathway, a pathway activated many human malignancies. The underlying mechanism of NFkB pathway protein interaction with the ZFTA-RELA fusion to drive tumorgenesis has not yet been described. In this study, we utilized our autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain to explore these questions. Using rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) targeting HA-tagged ZFTA-RELA fusion in our IUE model, we demonstrate that both canonical and non-canonical NFkB proteins interact with ZFTA-RELA protein in ZFTA-RELA fusion ependymoma. Focusing on select key NFkB proteins, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) targeting p50/NFkB1, a protein that partners with RELA/p65 in the canonical NFkB pathway, revealing that p50/NFkB1 regulatory programs define active genes that are both distinct from and shared with the ZFTA-RELA fusion protein. Functionally, we performed CRISPR-CAS9 knock out (KO) of p50/NFkB1 and identified no phenotypic effect, as KO lines demonstrated no growth impairment. Furthermore, NFkB targeting drugs did not impair tumor growth. Given these findings, suggesting redundant transcriptional programing, we identified that ZFTA and RELA intersect with TEAD transcription factor programs to activate YAP signaling and that p50 localizes to YAP-TEAD pathway members. Targeting TEAD 1 and 4 with ChIP-Seq, we found that TEAD 1 and 4 co-regulate p50/NFkB1 and ZFTA-RELA target genes. Finally, we demonstrate YAP pathway activation across all supratentorial ependymoma, including ZFTA-RELA ependymoma. Our pathway analysis consistently converged on YAP/TEAD pathways, suggesting collaboration of ZFTA-RELA fusion with YAP/TEAD in transcriptional regulation for these tumors. Targeting YAP/TEAD may therefore represent a promising therapeutic strategy for ZFTA-RELA fusion ependymoma.

BIOL-07. DISTINCTIVE FEATURES OF HIGH-GRADE GLIOMA MOUSE MODELS REVEALED BY SINGLE-NUCLEUS RNA-SEQUENCING GUIDE PRE-CLINICAL MODEL SELECTION

Abstract Glioma is the most common pediatric central nervous system tumor, with high-grade gliomas (HGG) having one of the worst prognoses of all human cancers. In order to develop better diagnostics and therapies, it is essential to use faithful disease models. Currently, highly passaged patient-derived xenograft (PDX) models are most widely used in preclinical studies, although alternative immunocompetent models are becoming increasingly available. Here, we compare several in vivo glioma models on a single-cell level and investigate their similarity to primary human tumors. Single-nucleus sequencing was used to analyze >125,000 nuclei of primary patient samples, xenografts, autochthonous mouse tumors and related allografts including early and late in vivo passages – with a focus on MET-fusion-driven as well as H3 K27M-mutant HGG. Transcriptomic profiles of single tumor cells and associated stromal/immune components reveal insights into model-specific intratumoral heterogeneity, tumor evolution, and similarities between mouse models and patient samples. In addition, matched tumors engrafted into immunocompromised and immunocompetent animals are used to examine tumor-immune crosstalk and the modifying role of the tumor microenvironment. This improved understanding of how the analyzed model systems evolve over time and how they reflect patient’s tumor compositions will allow to prioritize and refine modern preclinical trials.

Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.

Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

Targeted TLR9 Agonist Elicits Effective Antitumor Immunity against Spontaneously Arising Breast Tumors.

Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response. To model the clinical scenario of multiple tumor sites, we used MMTV-PyMT transgenic mice, which spontaneously develop heterogeneous breast tumors throughout their 10 mammary glands. We found that i.v. administration of a tumor-targeting TLR9 agonist, referred to as PIP-CpG, induced a systemic T cell-mediated immune response that not only promoted regression of existing mammary tumors, but also elicited immune memory capable of delaying growth of independent newly arising tumors. Within the tumor microenvironment, PIP-CpG therapy initiated an inflammatory cascade that dramatically amplified chemokine and cytokine production, prompted robust infiltration and expansion of innate and adaptive immune cells, and led to diverse and unexpected changes in immune phenotypes. This study demonstrates that effective systemic treatment of an autochthonous multisite tumor model can be achieved using a tumor-targeted immunostimulant and provides immunological insights that will inform future therapeutic strategies.

Melanocyte PDL1 promotes early tumor progression in a novel autochthonous NRAS-mutant melanoma model

Abstract Tumor-intrinsic PDL1 drives melanoma treatment resistance. Melanocytes (melanoma cell-of-origin) do not express PDL1, but PDL1 is expressed in malignant melanomas, suggesting melanocyte PDL1 promotes melanomagenesis. We developed a novel autochthonous mouse model that develops NRAS Q61R-mutant melanomas lacking PDL1 only in melanocytes (PDL1 KOTN Q61R) and littermate controls (PDL1 +/+TN Q61R). We induced mice with 4-OH tamoxifen ± UV exposure to accelerate melanoma development. Tumor latency significantly increased in PDL1 KOvs. PDL1 +/+TN Q61Rmice at 0 kJ/m 2and 2 kJ/m 2UV(p<0.02), suggesting melanocyte/tumor PDL1 promotes melanomagenesis. Latencies were similar at 4.5 kJ/m 2, suggesting immune contributions, e.g., from local immunosuppression. We derived transplantable cell lines from autochthonous tumors. PDL1 +/+TN Q61Rcell lines had increased mTORC1 and stemness, and reduced STING and reduced Chk1 inhibitor sensitivity in vitro and in vivo vs. PDL1 KOTN Q61R, phenocopied by PDL1 KOB16. Tumor PDL1 suppressed ERK signals (NRAS target) in TN Q61Rvs. PDL1 KOcell lines and promoted resistance to NRAS-targeting small molecule drugs, suggesting tumor PDL1 promotes NRAS signals. Transplanted 4.5 kJ/m 2UV-inducedPDL1 KOTN Q61Rtumors were resistant to αPD1, αPDL1, αPDL2, and CD122-biased IL2 in WT mice vs. B16 melanomas, which are sensitive to biased IL2. Our model allows studies of PDL1 signals in melanomagenesis and progression and distinguishes bona fide cell-intrinsic PDL1 signals. This research was funded by the NIH T32GM113896 (STX MSTP) Award (C.O.) and the NCI (CA204965, CA054515) supported Curiel.

FOXA2 Cooperates with Mutant KRAS to Drive Invasive Mucinous Adenocarcinoma of the Lung.

FOXA2 expression combined with mutant KRAS drives invasive mucinous adenocarcinoma of the lung by synergistically promoting a mucous transcriptional program, suggesting strategies for targeting this lung cancer type that lacks effective therapies.

Abstract 3: In vivo CRISPR activation screening identifies tissue-specific oncogene selection

Abstract Background: While the genetic landscapes of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) are relatively well described, the contribution of individual genes and transcriptomic programs to progression and metastasis remain incompletely understood. To quantify the tissue-specific selective advantage of transcriptomic drivers of tumor progression and metastasis, we have developed a platform for in vivo CRISPR activation (CRISPRa) oncogene screening. Material and Methods: Our platform consists of an autochthonous CRISPRa-competent mouse tumor model (PPKS; P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM), coupled with in vivo delivery of Cre/sgRNA lentiviral libraries. To evaluate tissue-specific differences in oncogene selection, we designed a CRISPRa library targeting 450 of the most frequently altered genes across human adenocarcinoma. We next generated lung and pancreas tumors through administration of controlled doses of Cre/sgRNA library in vivo. Tumors and metastatic lesions were isolated and guide enrichment was determined using NGS. We also performed CRISPRa screening on proto-oncogenic (PPKS) pancreatic organoids orthotopically transplanted into syngeneic or immunodeficient mice (NU/J). The results from our CRISPRa screening were confirmed using single-gene activation in vivo and in pancreatic organoids. Results: We found that pancreatic tumors displayed dramatically higher levels of guide selection relative to lung tumors. While, on average, 99.7% of guides were represented in primary lung tumors at survival endpoint, only 40.4% of guides were detected in pancreatic tumors. We identified very strong selection for Egfr-activation in primary lung tumors, followed by MafB, Sox2 and Myc. In contrast, we found near-complete dominance of Myc-activation in pancreatic tumors. Since EGFR and MYC represent two of the most frequently altered genes in NSCLC and PDAC, respectively, this indicates our ability to recapitulate clinically observed oncogene selection. To determine if the selection for Myc-activation in the pancreas was driven by immune response, we performed screening in pancreatic organoids transplanted into immune competent and incompetent mice and found that Myc was uniformly the most highly enriched target. To confirm our findings, we generated Myc-activated proto-oncogenic organoids and autochthonous tumors, and both displayed accelerated tumor formation relative to non-targeted controls. Conclusions: We present one of the first implementations of autochthonous in vivo CRISPR activation screening, allowing for direct observation and quantification of oncogene selection and competition in vivo. Our model faithfully recapitulated oncogene selection observed in patient tumors by identifying EGFR and MYC as dominating oncogenes in the context of KRAS mutant lung and pancreas tumors, respectively. Citation Format: Fredrik Ivar Thege, Sonja M. Woermann, Anirban Maitra. In vivo CRISPR activation screening identifies tissue-specific oncogene selection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3.

Abstract 1479: FOXA2 induces invasive mucinous adenocarcinoma of the lung in the presence of mutant KRAS

Abstract Introduction: The role of the endoderm lineage transcription factor FOXA2 in autochthonous lung tumors has been elusive though the expression of FOXA2 has been reported in human lung cancers. Here, using GEMM (Genetically Engineered Mouse Models) and human lung cancer cell lines, we delineate the role of FOXA2 in lung cancers. Experimental Procedures: GEMM that carry mutant KRAS or mutant EGFR along with FOXA2 (CCSP-rtTA; tetO-Foxa2; tetO-Kras.G12D or tetO-EGFR.L858R) were developed allowing for lung tumor induction by doxycycline administration. The growth of lung tumors affected by FOXA2 was assessed by microCT and pathologically. FOXA2 downstream genes were also evaluated in vivo by immunohistochemistry of mouse lung sections and in vitro by gain (ectopic lentivirus transduction) and loss (siRNA) of function approaches with human lung cancer cell lines using TaqMan gene expression, RNA-seq, ChIP-seq and CRISPRi analyses. FOXA2 protein expression in human lung cancer specimens was also assessed. Data: FOXA2 suppressed the growth of EGFR-mutant lung tumors while promoting that of KRAS-mutant lung tumors in GEMM. Notably, half of the KRAS-mutant lung tumors presented invasive mucinous adenocarcinoma of the lung (IMA) with the expression of a master mucous transcription factor SPDEF and the two major IMA mucin genes MUC5AC and MUC5B. In vitro, FOXA2 induced the expression of SPDEF, MUC5AC and MUC5B by directly binding to their loci. H3K27ac was introduced at the enhancer region of MUC5AC (most highly expressed IMA gene) by FOXA2 and the expression of MUC5AC initiated by FOXA2 was enhanced by mutant KRAS. FOXA2 was highly expressed in human IMA compared to normal lung. Conclusions: FOXA2 induces IMA in the presence of mutant KRAS by directly regulating the expression of mucous genes. Citation Format: Yutaka Maeda, Koichi Tomoshige, William D. Stuart, Iris M. Fink-Baldauf, Masaoki Ito, Tomoshi Tsuchiya, Takeshi Nagayasu, Tomoki Yamatsuji, Morihito Okada, Takuya Fukazawa, Minzhe Guo. FOXA2 induces invasive mucinous adenocarcinoma of the lung in the presence of mutant KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1479.

Abstract 3035: Phase I trial of alpha-lactalbumin vaccine in high-risk operable triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) has a poor prognosis and may be associated with germline mutations. α-lactalbumin (aLA) is expressed in lactating breasts but not at other times or in other tissues. Expression of aLA is found in 70% of TNBC (PMID: 27322324) so could be an immunologic target for TNBC based on the “retired protein hypothesis” (PMID: 31926646). In pre-clinical studies vaccination with aLA inhibited growth of established breast tumors and provided protection from development of autochthonous tumors in transgenic murine models of breast cancer and against 4T1 transplantable breast cancer in BALB/c mice (PMID: 20512124). Methods: To determine the safety and immunogenicity of aLA, patients with early stage TNBC are being entered in a Phase I trial of aLA with GMP-grade zymosan adjuvant in Montanide ISA 51 VG vehicle. Subjects receive 3 vaccinations given once every 2 weeks. Events of Common Terminology Criteria for Adverse Events (CTCAE) grade ≥2 are considered dose-limiting toxicities (DLTs). Results: CTCAE toxicity by dose level is summarized below. All DLTs were injection site reactions, with ulceration and need for incisional drainage representing the grade 3 events. ELISpot assays to determine frequencies of T-cells producing IFN-γ and IL-17 in response to recombinant aLA and ELISA assays of antibody response to aLA will be available in December 2022. Conclusion: Dose level 2 appears to be the maximum tolerated dose. Accrual to dose levels 1 and 2 will be expanded to further define toxicity and immunologic effects. Accrual of patients with BRCA1 or PALB2 mutations planning to undergo prophylactic mastectomy is beginning in order to define the toxicity and immunologic effects in this group and to determine whether inflammatory changes from occult lactational foci will be produced. Funding Source: Department of Defense (W81XWH-17-1-0592 and W81XWH-17-1-0593) Worst Toxicity by Dose Level Dose Level aLAC (mcg) dose Zymosan (mcg) dose n Patients n Grade 0 n Grade 1 n Grade 2 n Grade 3 1 10 10 3 3 2 100 10 3 3 3 500 10 3 2 1 Original 2 100 100 1 1 Citation Format: George Thomas Budd, Justin M. Johnson, Emily Rhoades, Halle Moore, Holly Levengood, Megan Kruse, Erin Roesch, Jame Abraham, Brenna Elliott, Elena Haury, Rachel Swartz, Holly Pederson, Zahraa Al Hilli, Vincent Tuohy. Phase I trial of alpha-lactalbumin vaccine in high-risk operable triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3035.

Genetically engineered PD-1 displaying nanovesicles for synergistic checkpoint blockades and chemo-metabolic therapy against non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) remains the most frequently diagnosed lung cancer and the leading cause of cancer-related mortality worldwide. PD-1/PD-L1 axis inhibitors have changed the treatment paradigm for various cancer types, including NSCLC. However, success of these inhibitors in lung cancer clinic is severely limited by their inability to inhibit the PD-1/PD-L1 signaling axis due to heavy glycosylation and heterogeneity expression of PD-L1 in NSCLC tumor tissue. Taking advantage of the facts that tumor cell derived nanovesicles could efficiently accumulate in the homotypic tumor sites due to their innate targeting abilities and that specific and high affinity existed between PD-1 and PD-L1, we developed NSCLC targeting biomimetic nanovesicles (NV) cargos from genetically engineered NSCLC cell lines that overexpressed PD-1 (P-NV). We showed that P-NVs efficiently bound NSCLC cells in vitro and targeted tumor nodules in vivo. We further loaded P-NVs with 2-deoxy-D-glucose (2-DG) and doxorubicin (DOX), and found that these drugs co-loaded P-NVs efficiently shrank lung cancers in mouse models for both allograft and autochthonous tumor. Mechanistically, drug-loaded P-NVs efficiently caused cytotoxicity to tumor cells and simultaneously activated anti-tumor immunity function of tumor-infiltrating T cells. Our data therefore strongly argue that 2-DG and DOX co-loaded, PD-1-displaying nanovesicles is a highly promising therapy for treatment of NSCLC in clinic. STATEMENT OF SIGNIFICANCE: Lung cancer cells overexpressing PD-1 are developed for preparing nanoparticles (P-NV). PD-1s displayed on NVs enhance their homologous targeting abilities to tumor cells expressing PD-L1s. Chemotherapeutics such as DOX and 2-DG, are packaged in such nanovesicles (PDG-NV). These nanovesicles efficiently delivered chemotherapeutics to tumor nodules specifically. The synergy between DOX and 2-DG is observed in inhibiting lung cancer cells in vitro and in vivo. Importantly, 2-DG causes deglycosylation and downregulation of PD-L1 on tumor cells while PD-1 displayed on nanovesicles' membrane blocks PD-L1 on tumor cells. 2-DG loaded nanoparticles thus activate anti-tumor activities of T cells in the tumor microenvironment. Our work thus highlights the promising antitumor activity of PDG-NVs, which warrants further clinical evaluation.

Phagocytosis increases an oxidative metabolic and immune suppressive signature in tumor macrophages.

Phagocytosis is a key macrophage function, but how phagocytosis shapes tumor-associated macrophage (TAM) phenotypes and heterogeneity in solid tumors remains unclear. Here, we utilized both syngeneic and novel autochthonous lung tumor models in which neoplastic cells express the fluorophore tdTomato (tdTom) to identify TAMs that have phagocytosed neoplastic cells in vivo. Phagocytic tdTompos TAMs upregulated antigen presentation and anti-inflammatory proteins, but downregulated classic proinflammatory effectors compared to tdTomneg TAMs. Single-cell transcriptomic profiling identified TAM subset-specific and common gene expression changes associated with phagocytosis. We uncover a phagocytic signature that is predominated by oxidative phosphorylation (OXPHOS), ribosomal, and metabolic genes, and this signature correlates with worse clinical outcome in human lung cancer. Expression of OXPHOS proteins, mitochondrial content, and functional utilization of OXPHOS were increased in tdTompos TAMs. tdTompos tumor dendritic cells also display similar metabolic changes. Our identification of phagocytic TAMs as a distinct myeloid cell state links phagocytosis of neoplastic cells in vivo with OXPHOS and tumor-promoting phenotypes.

Histone Deacetylase 6 Inhibition Exploits Selective Metabolic Vulnerabilities in LKB1 Mutant, KRAS Driven NSCLC

IntroductionIn KRAS-mutant NSCLC, co-occurring alterations in LKB1 confer a negative prognosis compared with other mutations such as TP53. LKB1 is a tumor suppressor that coordinates several signaling pathways in response to energetic stress. Our recent work on pharmacologic and genetic inhibition of histone deacetylase 6 (HDAC6) revealed the impaired activity of numerous enzymes involved in glycolysis. On the basis of these previous findings, we explored the therapeutic window for HDAC6 inhibition in metabolically-active KRAS-mutant lung tumors. MethodsUsing cell lines derived from mouse autochthonous tumors bearing the KRAS/LKB1 (KL) and KRAS/TP53 mutant genotypes to control for confounding germline and somatic mutations in human models, we characterize the metabolic phenotypes at baseline and in response to HDAC6 inhibition. The impact of HDAC6 inhibition was measured on cancer cell growth in vitro and on tumor growth in vivo. ResultsSurprisingly, KL-mutant cells revealed reduced levels of redox-sensitive cofactors at baseline. This is associated with increased sensitivity to pharmacologic HDAC6 inhibition with ACY-1215 and blunted ability to increase compensatory metabolism and buffer oxidative stress. Seeking synergistic metabolic combination treatments, we found enhanced cell killing and antitumor efficacy with glutaminase inhibition in KL lung cancer models in vitro and in vivo. ConclusionsExploring the differential metabolism of KL and KRAS/TP53-mutant NSCLC, we identified decreased metabolic reserve in KL-mutant tumors. HDAC6 inhibition exploited a therapeutic window in KL NSCLC on the basis of a diminished ability to compensate for impaired glycolysis, nominating a novel strategy for the treatment of KRAS-mutant NSCLC with co-occurring LKB1 mutations.

Abstract OT2-10-02: Phase I trial of an alpha-lactalbumin vaccine in patients with operable triple-negative breast cancer (TNBC)

Abstract Background: Triple-negative breast cancer (TNBC) is the subtype of breast cancer with the worst prognosis and is the subtype most often associated with germline mutations of BRCA1 as well as other genes. Alpha-lactalbumin (aLA) is a milk protein expressed in lactating breasts but not at other times or in other normal tissues. Expression of aLA is found in approximately 70% of TNBC (Cancers PMID: 27322324) so is an attractive immunologic target for TNBC based on the “retired protein hypothesis” (Semin Immunol PMID: 31926646). Pre-clinical studies have shown that vaccination with aLA inhibits the growth of established breast tumors and provides potent protection from development of autochthonous tumors in transgenic murine models of breast cancer and against 4T1 transplantable breast cancer in BALB/c mice (Nat Med PMID: 20512124). Specific Aims: We are conducting a Phase I trial of vaccination with an aLA vaccine in patients with early stage TNBC to demonstrate the safety of this approach and to document the ability to produce a relevant immunologic response to aLA. Trial Design: Patients are being entered into a Phase I trial of alpha-lactalbumin with GMP-grade zymosan adjuvant in Montanide ISA 51 VG vehicle. Subjects receive a total of 3 vaccinations administered once every 2 weeks. Toxic events of Common Terminology Criteria for Adverse Events (CTCAE) grade 2 or greater are considered dose-limiting. Dose levels being tested in the current protocol are alpha-lactalbumin/zymosan 0.01/0.01, 0.1/0.01, 0.5/0.01, 0.5/0.03, and 0.5 mg/0.06 mg. Patients are being monitored for toxicity until 84 days after the first vaccination or resolution of toxicity, whichever is later. Blood is being drawn prior to therapy and 14, 28, and 56 days after the first vaccination to assess cellular response using enzyme-linked immunosorbent spot (ELISpot) assays of interferon-gamma and interleukin-17 production in response to aLA. Humoral response to aLA vaccination is being assessed by enzyme-linked immunosorbent assay (ELISA). Eligibility Criteria: Patients with ER-negative, PR-negative, HER2-negative breast cancer of pathologic stage II-III or who had residual disease after standard pre-operative systemic therapy. Participants must be within 3 years of initiation of treatment and have no evidence of recurrence. Dose Selection: Doses are being escalated using a 3+3 trial design to determine the maximum tolerated dose, the lowest immunologic dose and the optimal immunologic dose Present Accrual and Target Accrual: Dependent on toxicity, 18-30 patients will be treated. Future Plans and Contact Information: After identification of the maximum tolerated dose and expansion of the dose levels associated with effective tumor immunity we plan to enroll 2 additional cohorts of patients: 1) patients who have completed pre-operative chemo-immunotherapy and are receiving pembrolizumab as standard of care therapy, and 2) patients without cancer planning to undergo prophylactic bilateral mastectomy. Interested parties may contact Dr. Budd at buddg@ccf.org. Funding Source: Department of Defense (W81XWH-17-1-0592 and W81XWH-17-1-0593) Citation Format: George Budd, Justin M. Johnson, Emily Rhoades, Halle Moore, Megan L. Kruse, Erin Roesch, Jame Abraham, Brenna Elliott, Elena Haury, Vincent K. Tuohy. Phase I trial of an alpha-lactalbumin vaccine in patients with operable triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-10-02.

A vector-encoded bispecific killer engager to harness virus-activated NK cells as anti-tumor effectors

Treatment with oncolytic measles vaccines (MV) elicits activation of immune cells, including natural killer (NK) cells. However, we found that MV-activated NK cells show only modest direct cytotoxic activity against tumor cells. To specifically direct NK cells towards tumor cells, we developed oncolytic measles vaccines encoding bispecific killer engagers (MV-BiKE) targeting CD16A on NK cells and carcinoembryonic antigen (CEA) as a model tumor antigen. MV-BiKE are only slightly attenuated compared to parental MV and mediate secretion of functional BiKE from infected tumor cells. We tested MV-BiKE activity in cocultures of colorectal or pancreatic cancer cells with primary human NK cells. MV-BiKE mediate expression of effector cytokines, degranulation and specific anti-tumor cytotoxicity by NK cells. Experiments with patient-derived pancreatic cancer cultures indicate that efficacy of MV-BiKE may vary between individual tumors with differential virus permissiveness. Remarkably, we confirmed MV-BiKE activity in primaryhuman colorectal carcinoma specimens with autochthonous tumor and NK cells.This study provides proof-of-concept for MV-BiKE as a novel immunovirotherapy to harness virus-activated NK cells as anti-tumor effectors.

Abstract IA007: Regulation of metastasis efficiency via mitochondrial genetics

Abstract While there has been increasing appreciation for the roles of mitochondria in cancer, most efforts investigating the genetics of cellular functions study only the nuclear genome. To overcome technical complications of studying the specific contributions of mitochondrial genetics, we created mitochondrial nuclear exchange (MNX) mice by transferring an oocyte nucleus from strain-x into an enucleated oocyte from strain-y. Using MNX mice, we made the following discoveries demonstrating the profound role(s) of mtDNA on several complex phenotypes. - Crossing nuclear genome-matched female mice with male transgenic mice bearing oncogenes, tumorigenicity and metastasis were up- or down-regulated depending upon mtDNA. - MNX mice exhibit different autochthonous tumor formation. - When syngeneic (i.e., histocompatible) mammary and melanoma cells were injected into MNX mice, metastasis changed 3-5X, depending on mtDNA SNP. C57BL/6J mtDNA inhibited metastasis while C3H/HeN mtDNA increased metastasis. - MNX mice have selectively altered epigenetic marks (DNA methylation and histone marks) in the nuclear genome. - MNX mice with higher ROS form more metastases. Scavenging ROS reduced metastasis. - Baseline immune profiles differ in MNX mice compared to nDNA-matched counterparts and polarization states of myeloid populations infiltrating lung metastases were also significantly different. - Metabolomic profiling of >5000 metabolites showed minor differences, none of which appear to correlate with the above-referenced phenotypic changes. - MNX mice have distinct gut microbiota than matched wild-type strains, suggesting mitochondrial-bacterial communication. Together, these results demonstrate that mitochondrial genes are among metastatic quantitative trait loci by intrinsic and extrinsic mechanisms. Candidate signals from mitochondria to the nucleus or bacteria will be reported. And since mitochondrial SNP are markers of genetic ancestry, our findings also suggest that mtDNA SNP could serve as prognostic biomarkers and partially explain racial disparities in disease severity. Support: Susan G. Komen for the Cure (SAC11037), METAvivor Research & Support Inc., National Foundation for Cancer Research, National Cancer Institute P30-CA168524. Citation Format: Danny R. Welch. Regulation of metastasis efficiency via mitochondrial genetics [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA007.

Abstract C024: CD206 small molecule activation recruits TLR1/2 to enhance cancer cell phagocytosis in reprogrammed tumor-associated macrophages and improves tumor control

Abstract Introduction: Targeting innate immune checkpoints on tumor-associated macrophages (TAMs) like CD40 activation or CD47 blockade is currently tested as a promising new therapeutic strategy in pancreas cancer. The mannose receptor CD206 is expressed on M2-like TAM populations and activation of CD206 can recalibrate these immune suppressive cells towards an anti-tumor M1-like phenotype to elicit effective innate immune responses and tumor control. The lack of a detailed understanding of the molecular mechanisms of CD206-mediated macrophage reprogramming, however, is an impediment for the design of novel, effective combination studies or the development of accurate biomarkers. Methods: Single-cell RNA-Seq analysis of TAM populations of autochthonous KP16 and KPC tumors treated with a CD206 checkpoint modulator was used to classify CD206 positive M2-like TAMs into responsive vs non-responsive to CD206 modulation. Candidates of differentially expressed innate immune regulators were evaluated for their role in CD206 activation in macrophages derived from transgenic knockout mouse models deficient for different innate immune regulators. The induction of CD206 protein-protein interactions with other innate immune regulators was examined via proximal ligation assays (PLA) or co-immunoprecipitation, as well as a series of CD206 mutants expressed in different reporter assays. CCR2-RFP transgenic mice were used to study cancer cell phagocytosis in vitro and in vivo. Results: CD206 activation via small molecule candidate NCGC00413972 induces a conformational change in CD206 which induces the formation of a TLR1/2-CD206 trimeric complex, activates SYK kinase, recruits MyD88, and activates NF-κB and type I interferon signaling. Carbohydrate recognition domain 3 (CRD3) of the non-activated CD206 receptor negatively regulates CD206-TLR1/2 interaction. CD206 small molecule ligation, or the deletion of the N-terminal CRDs of CD206, leads to the formation of active TLR1/2-CD206 complexes, NF-κB activation, and TAM reprogramming. Loss of SYK kinase binding sites in the cytoplasmatic domain of CD206 or inhibition of SYK abrogates NF-κB activation and reduces cell death of M2-like macrophages but does not affect interferon signaling or cancer cell phagocytosis. The combination of an FDA-approved SYK inhibitor with a CD206 small molecule activator increased the fraction of M2-to-M1 reprogrammed TAMs and cancer cell phagocytosis in the tumoral stroma of KPC mice. CD206 and TLR2 activators also cooperated in the reprogramming of TAMs, resulting in a recalibration of the immune landscape towards an anti-tumor phenotype and enhanced tumor control. Conclusions: Deconvolution of the mechanism of action of CD206 small molecule activation identifies toll-like receptor signaling as an essential co-regulator for the induction of innate immune responses. Co-stimulation of CD206 and TLR2 demonstrates therapeutic potential as an effective immunotherapy in a recalcitrant disease, as it cooperatively increases NF-κB signaling, cancer cell phagocytosis, and recalibrates myeloid cells. Citation Format: Sitanshu Sekhar Singh, Rushikesh V. Sable, Anju Kumari, Xin Hu, Abhijeet Kapoor, Raul Calvo, Emily Major, Taivan Odzorig, Laura Bassel, Serguei Kozolov, Mark Henderson, Juan Marugan, Udo Rudloff. CD206 small molecule activation recruits TLR1/2 to enhance cancer cell phagocytosis in reprogrammed tumor-associated macrophages and improves tumor control [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C024.

Abstract A067: Myc drives phenotypic heterogeneity, metastasis, and therapy resistance in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAc) ranks among the top three most aggressive cancers in the United States and is projected to increase in incidence over the next few years. Standard of care treatment for PDAc consists of a cocktail of harsh chemotherapies, which have improved overall survival by only a few percentage points—to a 5-year survival rate of 11%. One commonly deregulated pathway in PDAc is c-MYC (MYC), a potent transcription factor. MYC plays an important role in tumor progression and its deregulation has been correlated with tumor aggressiveness and therapeutic resistance in PDAc and other cancers. Recently, oncogenic MYC expression has been shown to regulate elements of the tumor microenvironment (TME) in mouse models of multiple cancers. In PDAc, MYC’s expression has been linked to a desmoplastic immune suppressive TME, yet the specific mechanism has yet to be described. Here, we present a novel genetically engineered mouse model (GEMM) of PDAc that can be used to better model the disease and to interrogate questions of how MYC regulates the tumor immune and stromal microenvironments. Our KMCERT2 model has inducible Cre-driven expression of both mutant Kras and low deregulated Myc in the pancreas. We show that deregulated MYC cooperates with KRASG12D in the adult pancreas to drive PDAc, and our model recapitulates inter- and intra-tumoral heterogeneity seen within clinical PDAc populations. Currently, a majority of murine studies of PDAc are performed using an embryonic KrasG12D- and p53 loss/mutant-driven PDAc model (KPC). RNA- and DNA sequencing on both microdissected autochthonous tumor specimens and KMCERT2 tumor-derived cell lines was conducted to further understand the mechanisms underlying our observed phenotypes. In contrast to the KPC model, our inducible KMCERT2 model of PDAc displays genetic changes, such as CDKN2A and SMAD4 loss, comparable to human disease. Interestingly, multiplexed immunohistochemistry analysis of immune cell composition of spontaneous KMCERT2 tumors compared to the commonly used KPC shows an increased density of antigen presenting cells (APCs) within MYC-driven tumors. Human PDAc is often resistant to standard of care therapies such as gemcitabine and FOLFIRINOX. Orthotopic therapeutic studies using our KMCERT2 tumor-derived cell lines demonstrate a similar resistance to these therapies, allowing us to use this model to better understand the mechanisms leading to therapeutic resistance and to test new therapies. In addition, we find consistent metastasis to the liver in both spontaneous and orthotopic transplant settings. Together, this work investigates the role of deregulated MYC expression in metastatic behavior, immune phenotypes, and therapeutic response in murine PDAc. It also provides both spontaneous and orthotopic mouse models of PDAc that recapitulate the heterogeneous and highly metastatic nature of the human disease, allowing for important therapeutic testing opportunities. Citation Format: Isabel A. English, Patrick J. Worth, Amy S. Farrell, Brittany L. Allen-Petersen, Vidhi Shah, Courtney Betts, Carl Pelz, Xiaoyan Wang, Colin J. Daniel, Mary C Thoma, Lisa M Coussens, Ellen M Langer, Rosalie C Sears. Myc drives phenotypic heterogeneity, metastasis, and therapy resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A067.

TGFβ control of immune responses in cancer: a holistic immuno-oncology perspective.

The immune system responds to cancer in two main ways. First, there are prewired responses involving myeloid cells, innate lymphocytes and innate-like adaptive lymphocytes that either reside in premalignant tissues or migrate directly to tumours, and second, there are antigen priming-dependent responses, in which adaptive lymphocytes are primed in secondary lymphoid organs before homing to tumours. Transforming growth factor-β (TGFβ) - one of the most potent and pleiotropic regulatory cytokines - controls almost every stage of the tumour-elicited immune response, from leukocyte development in primary lymphoid organs to their priming in secondary lymphoid organs and their effector functions in the tumour itself. The complexity of TGFβ-regulated immune cell circuitries, as well as the contextual roles of TGFβ signalling in cancer cells and tumour stromal cells, necessitates the use of rigorous experimental systems that closely recapitulate human cancer, such as autochthonous tumour models, to uncover the underlying immunobiology. The diverse functions ofTGFβ in healthy tissues further complicate the search for effective and safe cancer therapeutics targeting the TGFβ pathway. Here we discuss the contextual complexity of TGFβ signalling in tumour-elicited immune responses and explain how understanding this may guide the development of mechanism-based cancer immunotherapy.