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Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrate durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models, rep-resenting a variety of molecular and phenotypic subtypes of human melanomas and exhibiting their diverse range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify mechanisms and treatment strategies to improve patient care.

T cells play a central role in cancer immunosurveillance and current cancer immunotherapies, including adoptive cell transfer (ACT), T cell receptor or chimeric antigen receptor (CAR) T cell therapies and immune checkpoint blockade. Understanding the factors regulating T cell function is hence critical for improving the success of these immunotherapies. It has been recognized that metabolism can greatly affect different aspects of T cell function, including differentiation, cytokine production, longevity and exhaustion. Here we integrate genome-scale metabolic modeling (GEM) with biological experiments to discover novel metabolic determinants of T cell function. Previously we developed the metabolic transformation algorithm (MTA), a GEM method that was successfully applied to identify driving factors and targets for different diseases. Here applying MTA to data on CAR-T cell gene expression and patient response to anti-CD19 CAR-T therapy, we predicted mitochondrial metabolite transport and specifically proton transport in mitochondrial uncoupling, as key determinants of CAR-T therapy response. Mitochondrial uncoupling is also important for the in vivo persistence of adoptively transferred tumor-infiltrating lymphocytes, as further confirmed by analyzing their gene expressions from a KRAS-targeting ACT dataset. Focusing on the mitochondrial uncoupling protein 2 (Ucp2), which is abundantly expressed in T cells, we experimentally validated that it is required for T cell longevity and anti-tumor function. Specifically, the loss of Ucp2 either via knock-out (KO) or treatment by genipin (a Ucp2 inhibitor) in mice T cells results in accelerated differentiation into “terminal effector cells”, as shown by increased levels of T cell cytotoxicity and exhaustion markers, and decreased levels of central memory and stemness markers. Adoptive transfer of Ucp2-KO Pmel-1 T cells to mice bearing B16 melanomas displayed poorer anti-tumor efficacy and worse survival than the transfer of Ucp2-wildtype T cells. We find that Ucp2 modulates oxidative stress and DNA damage by regulating the levels of mitochondrial superoxide. Reducing mitochondrial reactive oxygen species was sufficient to rescue the loss of Ucp2-mediated effector T cell differentiation, senescence, cytokine production and anti-tumor activity Ucp2-KO Pmel-1 T cells. Tumor-specific CD8+ T cells could be metabolically reprogrammed by Ucp2 overexpression, which improved T cell longevity and anti-tumor function in the Pmel-1/B16 ACT mice model. In sum, our study establishes a novel role of Ucp2 in regulating T cell longevity and anti-tumor activity by repressing increased ROS levels accompanying mitochondrial dysfunction in differentiated and exhausted cells, suggesting that manipulating Ucp2 levels in T cells can be exploited to enhance T cell-based cancer immunotherapies. Citation Format: Madhusudhanan Sukumar, Kuoyuan Cheng, Arunakumar Gangaplara, Yogin Patel, Suman K. Vodnala, Rafiqul Islam, Arash Eidizadeh, Carolyn Subramaniam, Ping Lee, Rigel Kishton, Amanda N. Henning, Michael J. Kruhlak, Zhiya Yu, Ethan M. Shevach, Toren Finkel, Eytan Ruppin, Nicholas P. Restifo. Integrated computational and experimental analysis identifies the mitochondrial uncoupling protein 2 (Ucp2) as a key regulator of T cell anti-tumor function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1527.

Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrates durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models representing the main molecular and phenotypic subtypes of human melanomas and exhibiting their range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify new mechanisms and treatment strategies to improve patient care.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity., Author summary The majority of breast cancer-associated deaths are due to metastatic disease, the process where cancerous cells leave the primary tumor in the breast and spread to a new location in the body. To better understand the etiology of this process, we investigate the effects of gene expression changes in the primary tumor. In this study, we found that changing the expression of the gene Rnaseh2c changed the number of metastases that developed in the lungs of tumor-bearing mice. By investigating the enzyme complex Rnaseh2c is part of, RNase H2, we determined that Rnaseh2c’s effects may be independent of RNase H2 enzyme activity. Because Rnaseh2c is known to cause the autoimmune disease Aicardi-Goutières Syndrome (AGS), we tested whether the immune system is involved in the metastatic effect. Indeed, we found that the cytotoxic T cell response helps mediate the effect that Rnaseh2c has on metastasis. Together these data indicate that Rnaseh2c expression contributes to a patient’s susceptibility to developing breast cancer metastasis and demonstrate that the immune system is involved in this outcome. The implications of this study suggest immunotherapy could be a viable treatment for breast cancer metastasis and may help inform the understanding of AGS and RNase H2 in cancer.

Summary T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.

Cancer recurrence resulting from the metastatic outbreak of dormant disseminated tumor cells following the apparent successful treatment of the primary tumor is a major cause of breast cancer mortality. However, little is known regarding the molecular mechanisms governing tumour cell dormancy and the dormant-to-proliferative switch, impeding the development of effective therapeutic strategies. We therefore investigated whether stress-induced autophagy may promote survival of dormant cancer cells and, consequently, inhibition of autophagy could prevent breast cancer recurrence. To address the functional role of autophagy in breast cancer progression and the potential therapeutic impact of its inhibition, we utilized mouse and human 3D in vitro and in vivo preclinical models of dormancy. The analysis of autophagy markers and use of an autophagic flux biosensor allowed direct visualization of autophagic vesicles and their evolution in breast cancer dormant cells over time. In agreement with our hypothesis, pharmacologic or genetic inhibition of autophagy in dormant breast cancer cells resulted in significantly decreased cell survival and metastatic burden in vitro and in vivo. Furthermore, the inhibition of autophagy prevented the spontaneous dormant-to-proliferative switch of highly metastatic cells. In contrast, proliferating disseminated cells were insensitive to autophagy blockade. Indeed, in vivo analysis of the autophagic flux over time confirmed that autophagy is a critical survival process activated and maintained in dormant breast cancer cells, which is shut down after the cells undergo the dormant-to-proliferative switch. Transcriptomic analysis and in vivo metastatic burden assays identified the autophagy-related 7 (ATG7) gene, but not Beclin1 (BECN1), to be essential for autophagy activation, indicating that a non-canonical autophagy pathway is activated in dormant breast cancer cells. Co-localization studies identified mitochondria as the predominant autophagosomal cargo in breast cancer dormant cells. Mechanistically, inhibition of the autophagic flux in dormant breast cancer cells led to the accumulation of depolarized mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis. This study has important implications regarding the role of autophagy in breast cancer progression and suggests that inhibition of autophagy may be of therapeutic value in preventing breast cancer recurrence. Furthermore, it provides novel insights into the molecular mechanisms for survival of breast cancer dormant cells. Citation Format: Laura Vera Ramirez, Suman K. Vodnala, Ryan Nini, Kent W. Hunter, Jeffrey E. Green. Survival of dormant breast cancer cells and metastatic tumor recurrence is dependent upon the activation of autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 57.

Melanoma is an aggressive and lethal disease with no efficacious therapies for a broad subset of late stage patients. Current immunotherapies, including immune checkpoint blockade (ICB), may prolong survival in certain patients, but generate responses in less than 40% of the treated cohort. This demands a better understanding of molecular mechanisms underlying the lack of response and acquired resistance to ICB. However, functional studies are limited in patients, and current preclinical studies are handicapped by the absence of appropriate mouse models that recapitulate the pathological and immunological diversity of human melanomas. Here we develop four syngeneic melanoma mouse models with human-relevant genetic modifications and carcinogenic agents, which we hypothesize will mirror the spectrum of responses to ICB and offer a platform for future mechanistic studies in melanoma. The models are: 1) neonatal ultraviolet radiation (UV)-induced melanoma in a HGF-transgenic mouse, in which melanocyte localization at the epidermal-dermal junction mimics human distribution (HU); 2) 7,12-Dimethylbenz(a)anthracene (DMBA)-induced melanoma in a HGF-tg and Cdk4R24C mouse (HC4D); 3) UV-induced melanoma in a BrafCA/+; HGF-tg; Cdkn2aflox/+; Tyr-CreERT2-t mouse (BHCU); and 4) UV-induced melanoma in a BrafCA/+; Ptenflox/+; Cdkn2aflox/+; Tyr-CreERT2-tg mouse (BPCU). Exome sequencing of the four models reveals a high correlation with mutational subtypes previously described in human melanoma. BPCU and BHCU represent different Braf mutant patient populations and HU and HC4D represent triple wildtype melanoma (non-BRAF, -NRAS, -NF1). The four mouse models demonstrate distinct responses to ICB with anti-CTLA-4 treatment. While HU and HC4D melanomas show high or partial sensitivity to anti-CTLA-4, respectively, BPCU and BHCU do not respond to treatment. In vivo vaccination assays demonstrate that the anti-CTLA-4 response in our models is linked to increased tumor immunogenicity. However, the number of non-synonymous mutations and antigen presentation functionality do not correlate with ICB efficacy. Tumor infiltration by T cells was assessed by CD3 and FoxP3 immunostaining and gene expression analysis. Although clear differential gene expression profiles are noted among the four models and in those tumors responding to the treatment, we unexpectedly found that “hot” melanomas (e.g., showing upregulation of inflammatory pathways and high T-cell infiltration) do not necessarily predict ICB efficacy. These results suggest that additional mechanisms could help determine the response or intrinsic resistance to anti-CTLA-4 and open new avenues for future research and treatment. Overall, our study offers four genetically and phenotypically distinct mouse models representing diverse human melanoma subtypes as powerful tools for the mechanistic study of the response to immunotherapies in melanoma. Citation Format: Corinne Rauck, Eva Perez-Guijarro, Zoe W. Ohler, Rajaa El E. Meskini, Howard Yang, Suman Vodnala, Cari Graff-Cherry, Sung Chin, Anyen Fon, Helen Michael, Maxwell Lee, Terry Van Dyke, Shyam Sharan, Glenn Merlino, Chi-Ping Day. Developing a preclinical immunotherapy platform using syngeneic mouse models of human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5678.

Melanoma is the deadliest form of skin cancer due to the lack of widely effective therapies for advanced disease. Recently FDA-approved immunotherapies, such as immune checkpoint blockade (ICB) by CTLA-4 and PD-1/PD-L1 antibodies, provide unprecedent durable responses but in less than 40% of late stage melanoma patients. While high mutational loads characteristic of responsive tumors has not shown predictive value of patient outcome, accumulating evidences suggest a key role for neoantigens in the response to ICB. Moreover, new T-cell transfer- and vaccine-based therapeutic strategies highlighted the relevance of an efficient identification and prioritization of highly immunogenic neoantigens to improve immunotherapies. However, mechanistic studies are not possible in humans, and the development of adequate predictive methods is still the center of intense debate in the field. Here we use two genetically engineered melanoma mouse models exhibiting distinct response to ICB. Melanomas induced by neonatal ultraviolet radiation (UV) in a HGF-transgenic mouse (HGF-tg) showed high sensitivity to anti-CTLA-4, whereas UV-induced melanomas in HGF-tg; BrafV600E; Cdkn2a+/- mouse (Braf/HGF) did not respond. We hypothesized these models will allow us to identify the neoantigen features required for ICB response including type, expression levels and allele frequency patterns. High tumor immunogenicity, assessed by in vivo vaccination assays, as well as increased T-cell infiltration upon anti-CTLA-4 treatment were correlated to greater response. Moreover, exome and RNA sequencing analyses revealed similar mutational and neoantigen load in both models, albeit with no common expressed mutations. Notably, both models expressed similar levels of antigen presentation related genes (e.g. B2m, H2-Kd, Tap1) suggesting that specific neoantigens in HGF-tg melanoma cells may contribute to their sensitivity to anti-CTLA-4. To test this, we predicted MHC-I/-II binding of HGF-tg melanoma mutated epitopes in silico and generated a “neo-epitope” library. Importantly, the expression and allele frequency of most of selected mutations were decreased in anti-CTLA-4 responder HGF melanomas. The “neo-epitope” library was transduced into Braf/HGF non-responder cells and future studies will identify the neo-epitopes lost upon ICB, representing determinants of therapeutic success. Additionally, in vivo vaccination assays using synthetic mutant peptides will be performed to validate each neoantigen candidate. We anticipate that our studies will provide insight into the role that neoantigens play in melanoma immunotherapy responses. (EPG and CPD contribute to this abstract equally). Citation Format: Eva Perez Guijarro, Chi-Ping Day, Zoe W. Ohler, Rajaa El Meskini, Howard Yang, Suman Vodnala, Cari Graff-Cherry, Sung Chin, Anyen Fon, Helen Michael, Maxwell Lee, Terry Van Dyke, Shyam Sharan, Glenn Merlino. Functional characterization of neoantigens determining immune checkpoint blockade response in mouse models of human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5720.

Immune checkpoint blockers (ICBs) have rendered unprecedented, durable responses in metastatic melanoma, but the heterogeneous response among patients continues to be the major obstacle for their therapeutic development. It is generally hypothesized that neoantigens derived from mutated genes are involved in tumor response to ICBs, since the latter is correlated witt mutational loads of tumors. However, direct experimental evidence showing that threshold quantity or specific properties of neoantigens drive ICB response are mostly lacking. To identify and characterize neoantigens implicated in ICB response, we have generated two UV-induced melanoma models based on the BrafV600E/Pten-knockout (Braf/PKO) and Hgf-transgenic (Hgf-tg) mouse, which displayed intrinsic resistance and high sensitivity to an anti-CTLA-4 antibody, respectively. Exome sequencing identified 216 and 291 non-synonymous mutations in the Braf/PKO and UV-Hgf melanoma cell lines, respectively. By RNA sequencing, 74 (34%) and 121 (42%) of these mutated genes were found to be expressed in each model, respectively, and there were no overlapping mutations between them. The mutations found in the “sensitive” UV-Hgf melanoma were analyzed in silico for their binding affinity to MHC-I and/or MHC-II, thus characterizing putative neoantigens. A “neo-epitope” library was generated by cloning the DNA sequences flanking non-synonymous mutations in frame with the eGFP gene in a lentiviral vector. We further showed that such eGFP-fused epitopes can be presented by the cells to induce specific T cell responses. The library will be transduced into the “resistant” Braf/PKO melanoma, which will be treated with anti-CTLA-4 in mice to identify the neoantigens required for the response. To prevent immunity against eGFP expressed by tumors, the library-transduced melanoma cells will be transplanted into the eGFP-tolerant “glowing head mice”. The results will be used to determine if one, or more, of our candidate neo-epitopes can induce a response to anti-CTLA-4. We will also analyze if the response to this ICB is an epitope-specific reaction or require multiple epitopes, which will help to identify resistance mechanisms. We anticipate that our results will provide insight into the role of neoantigens in ICB response. Moreover, our models will serve as a platform to study the specific contribution and predictive value of neoantigens for melanoma response to immunotherapy, which could help improve therapeutic strategies involving ICBs. Citation Format: Chi-Ping Day, Eva Perez-Guijarro, Rajaa El Meskini, Zoe Weaver Ohler, Maxwell Lee, Howard Yang, Suman Vodnala, Shyam Sharan, Glenn Merlino. Identification of neo-antigens driving melanoma response to immune checkpoint blockers via in vivo screening [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 2623. doi:10.1158/1538-7445.AM2017-2623

Myeloid derived suppressor cells (MDSCs) are increased in tumor bearing condition, and elicit immunosuppression via type 2 polarization. We have previously reported that TGF-β signaling in MDSCs is essential for tumor metastasis. TGFβR2, a receptor essential for TGF-β signaling, is elevated in MDSCs. Deletion of Tgfbr2 in MDSCs, the gene encoding TGFβR2, significantly decreased tumor metastasis. However, it is unclear how TGFβR2 is regulated in MDSCs. We identify microRNA-130a and -145, which directly target Tgfbr2 in MDSCs and inhibit the expression of TGF-β receptor II (TβRII). Both miRs levels were lower in MDSCs from advanced tumor bearing mice correlating to increased TβRII level. Overexpression of miR-130a or -145 in MDSCs shift their immunological phenotype from type 2 to type 1 polarization. Moreover, tumor metastasis was significantly suppressed in myeloid specific miR-130 transgenic mice. In pre-clinical mouse models, the number of lung metastasis was significantly decreased when mice were treated with combination therapy of miR-130a or -145 with paclitaxel when compared with miR-130a, -145, or paclitaxel alone. We proposed that reprogramming of MDSCs by miR-130a and -145 could be a novel strategy in therapeutic treatment for breast cancer metastasis. Citation Format: Hiroki Ishii, Suman K. Vodnala, Bhagelu R. Achyut, Christine M. Hollander, Ashish Lal, Li Yang. miR-130a and -145 reprogram myeloid derived suppressor cells and enhance anti-tumor immunity [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 2944. doi:10.1158/1538-7445.AM2017-2944

Host immune surveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. Less is known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application of a T cell-dependent selection pressure in the form of a whole tumor vaccine would alter the immunogenic architecture in our transplantable murine melanoma model SB-3123. We generated an escape variant to test this hypothesis. We performed whole-exome and RNA-sequencing of the tumor line SB-3123 and two fresh tumors generated by subcutaneous implantation of SB-3123 to identify mutations and to assess levels of expression. There were 349 mutations shared between the tumor line and fresh tumors and less than 5 mutations were unique to each sample. We then investigated whether tumors that had undergone an additional round of host immunoediting would acquire different mutations. To test this, we generated a re-derived tumor cell line from a fresh SB-3123 tumor. Remarkably, the re-derived tissue culture line retained 333 mutations in common with the prior samples and produced only 2 unique mutations. To test the immunogenicity of SB-3123, we vaccinated mice with irradiated SB-3123 and administered a live tumor challenge two weeks after vaccination. This resulted in a profound vaccination response with mice being completely protected from tumor for over 200 days. However, one vaccinated mouse developed a recrudesced tumor at the site of implantation 40 days after challenge and a tumor line was created (SB-3123-esc). We asked whether SB-3123-esc represented an escape variant by immunizing mice with SB-3123 and then challenging with either SB-3123 or SB-3123-esc. Vaccination with SB-3123 did not protect against SB-3123-esc. We also performed sequencing on SB-3123-esc and discovered that 441 nonsynonymous mutations were common to both tumor lines while 80 were unique to SB-3123 and 40 to SB-3123-esc. Furthermore, we were able to determine that SB-3123-esc lost 2 immunogenic neoantigens but had acquired 5. In addition, we identified a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. Our results demonstrate that the expressed mutations of SB-3123 tumor did not change, regardless of whether we sequenced the tumor line, fresh tumors, or a re-derived tumor cell line. However, the escape tumor had new unique mutations and at least one of these was demonstrably immunogenic; we were able to develop a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. In conclusion, we posit that these observations offer a proof of concept that the process of cancer immune evasion can create new opportunities to treat escape tumors with T cell based therapies. Citation Format: Jenny H. Pan, Suman Vodnala, Robert L. Eil, Zhiya Yu, Jared Gartner, David Clever, Rahul Roychoudhuri, Shashank J. Patel, Christopher A. Klebanoff, Madhu Sukumar, Tori Yamamoto, Nicholas P. Restifo. Immunotherapy generates selective pressure to create an escape tumor with increased susceptibility to treatment with T cell based therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4034.

Meeting abstracts Host immunosurveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. It is less known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application

Tumor derived soluble factors result in accumulation of Gr-1+CD11b+ immature myeloid cells at tumor site and premetastatic organ. These cells are known to promote tumor progression and metastasis. Gr-1+CD11b+ immature myeloid cells and tumor-associated macrophages display a type 2 phenotype (M2) and produce immune suppressive cytokines including TGFβ. We have previously shown that immature myeloid cells are vicious producers of TGFβ ligand and express high levels of TGFβR2 receptor for eliciting immune suppressive function. Specific deletion of TGFβR2 in myeloid cells resulted in increased antitumor immunity and decreased metastasis in the lungs of cancer mouse models. microRNAs (miRs) are abundant class of small non-coding RNAs that have recently emerged as powerful epigenetic regulators of gene expression in pathophysiological conditions. One of the critical properties of miR is to target a large number of genes frequently within the context of a network, making them very efficient in regulating distinct biological cell processes. In the current project, we have identified three miRs that target myeloid TGFβR2 associated molecular network including p38 and cAMP pathways, which are critical in type 2 cytokine production. We have successfully transduced bone marrow stem cells with lentivirus expressing these miRs driven by a myeloid specific promoter CD11b. Transplantation of these modified bone marrow into lethally irradiated mice decreased breast cancer metastasis. The underlying mechanisms involve the reduction in type 2 cytokines such as IL-4, IL-10 and IL-13 and activation of IFN producing CD8 cytotoxic lymphocytes. Interestingly, the miRNAs are also differentially regulated by TGFβ signaling in TGFβR2+/+ and TGFβR2−/− myeloid cells, suggesting a feedback loop between TGFβ and miRNA. These results demonstrate that the candidate miRNA can reprogram tumor-associated myeloid cells by altering the cytokine milieu and enhance host antitumor immunity. Our work points out that endogenous miRNA targeting myeloid TGFβ signaling and its molecular network provides an important therapeutic option. Note: This abstract was not presented at the meeting. Citation Format: Suman K. Vodnala. MicroRNA-mediated reprogramming of myeloid cells by targeting TGFβ signaling and its associated molecular network. [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 3128. doi:10.1158/1538-7445.AM2015-3128