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Every cell in the human body has a unique set of somatic mutations, yet it remains difficult to comprehensively genotype an individual cell1. Here, we developed solutions to overcome this obstacle in the context of normal human skin, thus offering the first glimpse into the genomic landscapes of individual melanocytes from human skin. As expected, sun-shielded melanocytes had fewer mutations than sun-exposed melanocytes. However, within sun-exposed sites, melanocytes on chronically sun-exposed skin (e.g. the face) displayed a lower mutation burden than melanocytes on intermittently sun-exposed skin (e.g. the back). Melanocytes located adjacent to a skin cancer had higher mutation burdens than melanocytes from donors without skin cancer, implying that the mutation burden of normal skin can be harnessed to measure cumulative sun damage and skin cancer risk. Moreover, melanocytes from healthy skin commonly harbor pathogenic mutations, though these mutations tended to be weakly oncogenic, likely explaining why they did not give rise to discernible lesions. Phylogenetic analyses identified groups of related melanocytes, suggesting that melanocytes spread throughout skin as fields of clonally related cells, invisible to the naked eye. Overall, our study offers an unprecedented view into the genomic landscapes of individual melanocytes, revealing key insights into the causes and origins of melanoma.

Cellular heterogeneity is a major cause of treatment resistance in cancer. Despite recent advances in single-cell genomic and transcriptomic sequencing, it remains difficult to relate measured molecular profiles to the cellular activities underlying cancer. Here, we present an integrated experimental system that connects single cell gene expression to heterogeneous cancer cell growth, metastasis, and treatment response. Our system integrates single cell transcriptome profiling with DNA barcode based clonal tracking in patient-derived xenograft models. We show that leukemia cells exhibiting unique gene expression signatures respond to different chemotherapies in distinct but consistent manners across multiple mice. In addition, we uncover an unexpected yet common form of leukemia expansion that is spatially confined to the bone marrow of single anatomical sites and driven by cells with distinct gene expression signatures. Our integrated system directly and effectively interrogates the molecular and cellular basis of the intratumoral heterogeneity underlying disease progression and treatment resistance.

Background Checkpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME). Methods We developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI. Results Using a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs. Conclusion Taken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer. Statement of significance CPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.

BackgroundCheckpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME).MethodsWe developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI.ResultsUsing a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs.ConclusionTaken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer.Statement of significanceCPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.

Every cell in the human body has a unique set of somatic mutations, yet it remains difficult to comprehensively genotype an individual cell. Here, we developed solutions to overcome this obstacle in the context of normal human skin, thus offering the first glimpse into the genomic landscapes of individual melanocytes from human skin. We comprehensively genotyped 133 melanocytes from 19 sites across 6 donors. As expected, sun-shielded melanocytes had fewer mutations than sun-exposed melanocytes. However, within sun-exposed sites, melanocytes on chronically sun-exposed skin (e.g. the face) displayed a lower mutation burden than melanocytes on intermittently sun-exposed skin (e.g. the back). Melanocytes located adjacent to a skin cancer had higher mutation burdens than melanocytes from donors without skin cancer, implying that the mutation burden of normal skin can be harnessed to measure cumulative sun damage and skin cancer risk. Moreover, melanocytes from healthy skin commonly harbor pathogenic mutations, likely explaining the origins of the melanomas that arise in the absence of a pre-existing nevus. Phylogenetic analyses identified groups of related melanocytes, suggesting that melanocytes spread throughout skin as fields of clonally related cells, invisible to the naked eye. Overall, our study offers an unprecedented view into the genomic landscapes of individual melanocytes, revealing key insights into the causes and origins of melanoma.

Introduction: Chimeric antigen receptor (CAR) T cell immunotherapy has had only modest success in solid malignancies due in part to tumor antigen heterogeneity and the immunosuppressive tumor microenvironment. We have previously demonstrated that administration of cyclophosphamide (CTX) prior to CAR T infusion promoted the eradication of solid tumors that lacked homogenous expression of the CAR target antigen. Although CTX has multiple effects, we hypothesized that one potential mechanism was depletion of T-regulatory cells (Treg). To test this, we used a genetic model of Treg depletion involving mice with FOXP3-driven diphtheria toxin receptor expression (FOXP3-DTR) and an orally administered chemokine receptor 4 (CCR4) antagonist called CCR4-351. Treg express CCR4 and migrate toward CCL17 and CCL22 which are frequently upregulated by tumors. We have previously shown that the tool compound CCR4-351 decreases Treg accumulation in tumors. Methods: Transgenic FOXP3-DTR C57BL/6 mice were injected s.c. in the flank with a syngeneic murine mesothelioma cell line expressing human mesothelin. When tumors reached ~85 mm3, diphtheria toxin was given i.p. and 24 hours later, 2 doses of 107 transduced human anti-mesothelin CAR T cells (M11) were injected i.v. 2 days apart. In our pharmacologic study, wild type, tumor-bearing C57BL/6 mice were given CCR4-351 daily at 50 mg/kg by oral gavage. Mice were treated with M11 CAR T cells as above, starting one day after CCR4-351 therapy. Tumor growth and mouse survival were determined. Results: Genetic depletion of Treg alone did not significantly slow tumor growth compared to untreated control. However, the combination of M11 CAR T cells plus genetic depletion of Treg significantly slowed tumor growth compared to vehicle control (p Conclusions: Treg depletion via systemic ablation or inhibition of trafficking, resulted in augmentation of the antitumor effects of CAR T cells in our pre-clinical immunocompetent solid tumor model. If generalizable, these data support further investigation as to whether the orally bioavailable CCR4 antagonist currently in clinical development, FLX475, may be used to augment human CAR T cell therapy for solid tumors. Citation Format: Michael S. Leibowitz, Nicholas S. Olimpo, Liqing Wang, Aparna Jorapur, Deepa Pookot, Maria Liousia, Evguenia Arguiri, Jing Sun, Astero Klampatsa, Dirk G. Brockstedt, Wayne W. Hancock, Steven M. Albelda. T-regulatory cells impair CAR T cell-mediated antitumor activity in a murine solid tumor model [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 1585.

Digital holographic cytometry (DHC) permits label-free visualization of adherent cells. Dozens of cellular features can be derived from segmentation of hologram-derived images. However, the accuracy of single cell classification by these features remains limited for most applications, and lack of standardization metrics has hindered independent experimental comparison and validation. Here we identify twenty-six DHC-derived features that provide biologically independent information across a variety of mammalian cell state transitions. When trained on these features, machine-learning algorithms achieve blind single cell classification with up to 95% accuracy. Using classification accuracy to guide platform optimization, we develop methods to standardize holograms for the purpose of kinetic single cell cytometry. Applying our approach to human melanoma cells treated with a panel of cancer therapeutics, we track dynamic changes in cellular behavior and cell state over time. We provide the methods and computational tools for optimizing DHC for kinetic single adherent cell classification.

EPHB4, an ephrin type B receptor, is implicated in the growth of several epithelial tumors and is a promising target in cancer therapy; however, little is known about its role in hematologic malignancies. In this article, we show that EPHB4 is highly expressed in ∼30% of acute myeloid leukemia (AML) samples. In an unbiased RNA interference screen of primary leukemia samples, we found that EPHB4 drives survival in a subset of AML cases. Knockdown of EPHB4 inhibits phosphatidylinositol 3-kinase/AKT signaling, and this is accompanied by a reduction in cell viability, which can be rescued by a constitutively active form of AKT. Finally, targeting EPHB4 with a highly specific monoclonal antibody (MAb131) is effective against AML in vitro and in vivo. EPHB4 is therefore a potential target in AML with high EPHB4 expression.

Loss of the CDKN2A tumor suppressor is associated with melanoma metastasis, but the mechanisms connecting the phenomena are unknown. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we discovered that a lineage restricted transcription factor, BRN2, is downstream of CDKN2A and directly regulated by E2F1. In a cohort of melanocytic tumors that capture distinct progression stages, we observed that CDKN2A loss coincides with both the onset of invasive behavior and increased BRN2 expression. Loss of the CDKN2A protein product p16(INK4A) permitted metastatic dissemination of human melanoma lines in mice, a phenotype rescued by inhibition of BRN2. These results demonstrate a mechanism by which CDKN2A suppresses the initiation of melanoma invasion through inhibition of BRN2.

Individual cells within isogenic tumor populations can exhibit distinct cellular morphologies, behaviors, and molecular profiles. Cell state plasticity refers to the propensity of a cell to transition between these different morphologies and behaviors. Elevation of cell state plasticity is thought to contribute to critical stages in tumor evolution, including metastatic dissemination and acquisition of therapeutic resistance. However, methods for quantifying general plasticity in mammalian cells remain limited. Working with a HoloMonitor M4 digital holographic cytometry platform, we have established a machine learning-based pipeline for high accuracy and label-free classification of adherent cells. We use twenty-six morphological and optical density-derived features for label-free identification of cell state in heterogeneous cultures. The system is housed completely within a mammalian cell incubator, permitting the monitoring of changes in cell state over time. Here we present an application of our approach for studying cell state plasticity. Human melanoma cell lines of known metastatic potential were monitored in standard growth conditions. The rate of feature change was quantified for each individual cell in the populations. We observed that cells of higher metastatic potential exhibited more rapid fluctuation of cell state in homeostatic conditions. The approach we demonstrate will be advantageous for further investigations into the factors that influence cell state plasticity.

Deep penetrating nevus (DPN) is characterized by enlarged, pigmented melanocytes that extend through the dermis. DPN can be difficult to distinguish from melanoma but rarely displays aggressive biological behavior. Here, we identify a combination of mutations of the β-catenin and mitogen-activated protein kinase pathways as characteristic of DPN. Mutations of the β-catenin pathway change the phenotype of a common nevus with BRAF mutation into that of DPN, with increased pigmentation, cell volume and nuclear cyclin D1 levels. Our results suggest that constitutive β-catenin pathway activation promotes tumorigenesis by overriding dependencies on the microenvironment that constrain proliferation of common nevi. In melanoma that arose from DPN we find additional oncogenic alterations. We identify DPN as an intermediate stage in the step-wise progression from nevus to melanoma. In summary, we delineate specific genetic alterations and their sequential order, information that can assist in the diagnostic classification and grading of these distinctive neoplasms., Deep penetrating nevi (DPN) are unusual melanocytic neoplasms with unknown genetic drivers. Here the authors show that majority of DPN harbor activating mutations in the β-catenin and the MAP-kinase pathways; this characteristic can help in the classification and grading of these distinctive neoplasms.

Digital holographic microscopy permits live and label-free visualization of adherent cells. Here we report the application of this approach for high accuracy kinetic quantitative cytometry. We identify twenty-six label-free optical and morphological features that are biologically independent. When used as a basis for machine learning, these features allow blind single cell classification with up to 95% accuracy. We present methods to control for inherent holographic noise, thereby establishing a set of reliable quantitative features. Together, these contributions permit continuous digital holographic cytometry for three or more days. Applying our approach to human melanoma cells treated with a panel of cancer therapeutics, we can track the response of each cell, simultaneously classifying multiple behaviors such as cell cycle length, motility, apoptosis, senescence, and heterogeneity of response to each therapeutic. Importantly, we demonstrate relationships between these phenotypes over time. This work thus provides an experimental and computational roadmap for low cost live-cell imaging and kinetic classification of heterogeneous adherent cell populations.

The tumor microenvironment (TME) is characterized by deficiencies in oxygen and key nutrients, such as glucose and amino acids, resulting in an overall immune-suppressive environment. Key suppressive cell types in the TME include tumor, stromal and myeloid-derived suppressor cells (MDSC) which create a nutrient-poor environment that supports tumor growth and limits immune surveillance. General control nonderepressible 2 (GCN2), a stress response kinase, plays a key role in sensing and modulating the cellular response to amino acid deprivation. GCN2 activation in T cells triggers the integrated stress response pathway and promotes T cell anergy and apoptosis. We have developed small molecule GCN2 inhibitors (GCN2i) that are highly potent and selective in vitro. Culturing primary mouse or human immune cells under low nutrient conditions activates the GCN2 pathway limiting T cell proliferation and function. Treatment of these nutrient-deprived T cells with GCN2i resulted in rescue of CD8+ T cell proliferation and effector functions. In addition, GCN2 inhibition in MDSC alone fully reversed CD33+MDSC-induced T cell suppression and effector functions. Our GCN2 inhibitors are orally bioavailable with drug like in vivo ADME properties. Our GCN2i is currently being evaluated in vivo, in murine syngeneic tumor models. Our results demonstrate that inhibition of GCN2 is an attractive approach for relieving T cell suppression and promoting effector function, demonstrating GCN2 as a promising therapeutic target for the treatment of cancer. Note: This abstract was not presented at the meeting. Citation Format: Buvana Ravishankar, Lavanya Adusumilli, Deepa Pookot Pookot, Emily Huang, Raashi Sreenivasan, Lisa Marshall, Deepika Kaveri, Oezcan Talay, Silpa Suthram, Svetlana Miakicheva, Abood Okal, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Parcharee Tivitmahaisoon, Scott Jacobson, Steve Wong, Angela Wadsworth, Jerick Sanchez, Martin Brovarney, David Chian, Sachie Marubayashi, Aparna Jorapur, Delia Bradford, Christophe Colas, Gene Cutler, Jacob Schwartz, David Wustrow, Paul Kassner, Dirk Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1104.

Type 2 helper T cells (Th2)cells have been shown to express CCR4 receptor, and play a critical role in driving the pathogenesis of asthma and atopic dermatitis. FLX193 is a best-in-class, highly-potent and selective small molecule CCR4 antagonist under investigation for the treatment of allergic disorders. FLX193 blocked migration of CCR4+ Th2 cells (human and mouse) towards CCL17 and CCL22 in an in vitro chemotaxis assay. FLX193 is well-tolerated in animals at efficacious doses. In an Ovalbumin (OVA)-induced asthma model, FLX193 significantly reduced lymphocyte and eosinophil counts in the Bronchoalveolar lavage (BAL) fluid and showed a reduction of the effector Th2-relevant cytokines IL-5 and IL-13. FLX193 treatment also reduced the levels of CCL17 and CCL22 in the BAL fluid, indicating an overall reduction of inflammation. In addition, we used an atopic dermatitis mouse model to demonstrate that treatment with FLX193 decreased CCR4+ T-cell mediated inflammation. Hence, FLX193 shows promise in the treatment of atopic dermatitis.

CDKN2A acts as a critical tumor suppressor in melanoma, as evidenced by frequent loss of function mutations and deletion. Loss of CDKN2A is believed to permit escape from senescent pre-neoplastic cell populations through relieve of a cell cycle block mediated by its two gene products. We performed a comprehensive analysis of CDKN2A gene status, mRNA and protein expression levels of p16 and p14 in a cohort of melanomas and their adjacent pre-neoplastic lesions and observed that bi-allelic CDKN2A loss coincides with the progression stage when primary melanomas become invasive. In melanoma lines, p16INK4A, one of the protein products of the CDKN2A locus, is a potent barrier to metastasis, independent of its known role inhibiting cell proliferation. We genetically engineered primary human melanocytes to harbor CDKN2A deletions and/or BRAF V600E mutation at their endogenous BRAF locus. Using this physiologic model for the early phases of neoplastic transformation, we found no evidence for BRAF-induced senescence, rather observing that p16INK4A loss activates a master regulator of melanoma invasion, BRN2, through Rb-E2F1 pathway. These results demonstrate that one of the most frequently altered genes across human cancers, CDKN2A, has an unexpected novel role in inhibiting cellular invasion through lineage specific transcription factors and acts as an essential gatekeeper of early metastatic dissemination. Citation Format: Hanlin Zeng, Aparna Jorapur, A. Hunter Shain, Ursula E. Lang, Rodrigo Torres, Yuntian Zhang, Thomas Botton, Jue Lin, Andrew S. Mcneal, Matthew Donne, Ingmar N. Bastian, Jeffrey North, Laura Pincus, Richard Yu, Beth S. Ruben, Nancy Joseph, Iwei Ye, Boris C. Bastian, Robert L. Judson. Bi-allelic loss of CDKN2A initiates melanoma invasion and metastasis via E2F1-BRN2 axis [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 5518.

We have performed experiments to test whether Epstein Barr Virus (EBV)-infected tumors are enhanced for regulatory T cell (Treg) infiltration and whether selective and potent CCR4 antagonists would be a particularly effective therapeutic in this class of indications. Treg cells, which contribute to an immune-suppressive tumor microenvironment (TME), are attracted to tumors via the recognition of CCL17 and CCL22 ligands by the CCR4 receptor. These chemokines have been shown to be expressed in cells infected by the Epstein Barr Virus (EBV) via the viral LMP1 gene. Tumor types which are frequently associated with EBV-infection include gastric adenocarcinoma (~10% positive), classical Hodgkin's Lymphoma (~50%), and nasopharyngeal carcinoma (~100%). Analyzing RNA expression in EBV-associated tumors, we found strong expression of CCL17, CCL22, and FOXP3, a marker of Treg, when compared to EBV-negative tumors. In fact, NPC tumors show extremely high FOXP3 levels. To further test this link, we obtained EBV-associated tumor samples and performed RNA in situ hybridization (ISH) to measure co-expression of these genes. Strong co-localization, was indeed found, further supporting a link between EBV and Treg recruitment. To directly test whether EBV-positive tumors recruit Treg into tumors via CCL22/17 upregulation, we developed models in which mice were inoculated subcutaneously with EBV-positive cancer cell lines. These EBV-positive cancer cells were assessed for chemokine production in vitro and in vivo by ELISA. We assessed tumor-infiltrating lymphocytes (TILs) in established tumors, including Treg, CD4+ and CD8+ T cells as well as T cell activation markers. Treating these tumor-bearing mice with selective and potent CCR4 small-molecule antagonists alone or in combination with checkpoint-targeting antibodies allowed us to demonstrate meaningful antitumor responses in EBV-positive tumors. Together, these data suggest that EBV-positive tumors, such as gastric adenocarcinomas, Hodgkin's lymphomas, and nasopharyngeal carcinomas, are a class of indications of particular interest and potentially increased responsiveness to small-molecule CCR4 antagonists. These results are helping to inform the ongoing FLX475 trials currently in the clinic. Citation Format: Oezcan Talay, Aparna Jorapur, Scott Jacobson, Sachie Marubayashi, Lisa Marshall, Silpa Suthram, Omar Robles, John Ketcham, Maureen K. Reilly, Ashkaan Younai, Berenger Biannic, Dennis Hu, Minna Bui, Jacob Schwarz, Paul Kassner, Gene Cutler. EBV associated tumors have increased regulatory T cell recruitment and are therefore a potential indication for treatment with potent and selective small molecule CCR4 antagonists [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 4752.

The Hedgehog (Hh) signaling pathway plays a critical role in embryonic development and adult tissue homeostasis and has emerged as an important therapeutic target in many cancers, including leukemia and myeloproliferative diseases. Our mechanistic understanding of Hh pathway signaling and regulation comes primarily from developmental studies in neural and limb development. Studies of Hedgehog signaling in the hematopoietic system have produced contradictory results, and no clear consensus regarding Hh signaling in normal hematopoiesis is available to inform the role of Hedgehog signaling in hematologic malignancies. In our work we have focused on understanding the downstream effectors of Hedgehog signaling, the Gli transcription factors. The three Gli proteins, Gli1, Gli2 and Gli3 have both transcriptional activator and repressor functions, which allow for regulation and fine-tuning of Hedgehog pathway output. Previous studies from our group have revealed that Gli1null HSCs had no defects in self-renewal, however myeloid differentiation and stress hematopoiesis were severely impaired (Merchant, et al., Blood 2010). In normal tissues, Hh pathway activation via Ptch/Smo causes an increase in the downstream activating transcription factor GLI1 and a decrease in the transcriptional repressor Gli3R. Our recent studies demonstrated that GLI3R has a tumor suppressor role in human acute myeloid leukemia by directly repressing AKT expression (Chaudhry et al., AACR Annual Meeting 2015). To date nothing is known about the role of Gli3 in normal hematopoiesis. In the present study, we crossed Vav-Cre transgenic mice to Gli3fl/fl mice to generate mice with a conditional loss of Gli3 (Gli3null) in the hematopoietic system. HSC self-renewal was analyzed by serial transplant. In comparison to HSCs from Gli3 wild type (Gli3WT) mice bone marrow (BM), HSCs from Gli3null BM showed decreased long-term engraftment and self-renewal. In addition, quantification of long-term HSC (LT-HSC, CD34neg Flt3neg KSL), short-term HSC (ST-HSC, CD34+ Flt3neg KSL), and multi-potent progenitor (MPP, CD34+ Flt3+ KSL) revealed that the frequency of LT-HSCs in Gli3null BM (0.004-0.007%) was lower compared to Gli3 WT BM (0.008-0.02%). In mice transplanted with Gli3null BM, myeloid expansion was observed with a block in T and B cell lineage differentiation. Analysis of the c-Kit+ Sca1neg Linneg (KL) myeloid progenitor compartment revealed a two-fold increase in the FcRγhigh CD34+ KL granulocyte-monocyte progenitors (GMPs) in Gli3null BM, suggesting an expansion of granulocytic compartment. Since Gli3R is a key negative regulator of Gli1, these are consistent with decrease in GMP and myeloid differentiation previously seen in Gli1null mice. In summary, our studies reveal a previously unknown function for Gli3 in regulating HSCs and myeloid differentiation, and help to elucidate the complex regulation of Hh signaling in the hematopoietic system. Disclosures No relevant conflicts of interest to declare.

The Hedgehog (Hh) signaling pathway is activated in most hematological and solid cancers and is a promising target for therapeutic development. Deletions in the receptor Patched (PTCH) or activating mutations in the upstream positive regulator Smoothened (SMO) have been reported in a few cancers such as basal cell carcinoma and medulloblastoma, but are largely absent in most tumor types; therefore, the mechanism of pathway activation in most cancers remains unknown. In normal tissues, Hh pathway activation via PTCH/SMO causes an increase in the downstream activating transcription factor GLI1 and a decrease in the transcriptional repressor GLI3R. We confirm that the Hh pathway is active in acute myeloid leukemia (AML) and is required for cell survival, however, this activity is largely independent of the upstream activator SMO. Using siRNA knock down of GLI1, GLI3 and SMO in AML cell lines, Gli3 conditional knock out mice, and primary AML samples, we are able to show that loss of the GLI3R repressor function is sufficient to activate Hh target gene expression independent of ligand binding, and GLI3R levels correlate inversely with GLI1 levels. Importantly, we show that GLI3R is required for the therapeutic effect of SMO antagonists in AML samples and restoration of GLI3R restores sensitivity to SMO antagonists in AML cell lines. Epigenetic and gene expression analysis of the TCGA AML data set reveals that the GLI3 expression is silenced in most AML patient samples. We demonstrate in vitro and in patients that treatment with hypomethylating agents restores GLI3R levels and sensitivity to SMO antagonists in AML. In a transgenic mouse model of myeloproliferative disease (vav-Jak2V617F, gift of Joe Zhao, OUHSC) effective treatment with SMO antagonists are associated with increases in GLI3R but no changes in GLI1. Finally we show that GLI3R can directly repress expression of AKT and in vivo responses to SMO antagonists are correlated with changes in AKT expression, independent of GLI1 levels. In summary, this study provides the first evidence that GLI3R plays an essential role in SMO independent Hh signaling in AML, and suggests that GLI3R could serve as a potential biomarker for patient selection in SMO antagonist clinical trials. Furthermore, these data provide important mechanistic data to support rational combinations of hypomethylating agents with SMO antagonists in clinical trials. Citation Format: Parvesh Chaudhry, Mohan Singh, Tim Triche, Aparna Jorapur, Parkash S. Gill, Akil Merchant. GLI3 repressor levels determine Hedgehog pathway activity and predict response to Smoothened antagonist in acute myeloid leukemia. [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 2090. doi:10.1158/1538-7445.AM2015-2090