A multi-omic single cell sequencing approach to develop a CD8 T cell specific gene signature for anti-PD1 response in solid tumors.

Immune checkpoint blockade (ICB) has led to durable clinical responses in multiple cancer types. However, biomarkers that identify which patients are most likely to respond to ICB are not well defined. Many putative biomarkers developed from a small number of samples often fail to maintain their predictive status in larger validation cohorts. We show across multiple human malignancies and syngeneic murine tumor models that neither pretreatment T cell receptor (TCR) clonality nor changes in clonality after ICB correlate with response. Dissection of tumor infiltrating lymphocytes pre- and post-ICB by paired single-cell RNA sequencing and single-cell TCR sequencing reveals conserved and distinct transcriptomic features in expanded TCR clonotypes between anti-PD1 responder and nonresponder murine tumor models. Overall, our results indicate a productive anti-tumor response is agnostic of TCR clonal expansion. Further, we used single-cell transcriptomics to develop a CD8+ T cell specific gene signature for a productive anti-tumor response and show the response signature to be associated with overall survival (OS) on nivolumab monotherapy in CheckMate-067, a phase 3 clinical trial in metastatic melanoma. These results highlight the value of leveraging single-cell assays to dissect heterogeneous tumor and immune subsets and define cell-type specific transcriptomic biomarkers of ICB response.

Critical role of kinase activity of hematopoietic progenitor kinase 1 in anti-tumor immune surveillance.

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of-function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens.

Discovery of 4-Azaindole Inhibitors of TGFßRI as Immuno-oncology Agents.

The multifunctional cytokine TGFß plays a central role in regulating antitumor immunity. It has been postulated that inhibition of TGFß signaling in concert with checkpoint blockade will provide improved and durable immune response against tumors. Herein, we describe a novel series of 4-azaindole TGFß receptor kinase inhibitors with excellent selectivity for TGFß receptor 1 kinase. The combination of compound 3f and an antimouse-PD-1 antibody demonstrated significantly improved antitumor efficacy compared to either treatment alone in a murine tumor model.

Heterobicyclic inhibitors of transforming growth factor beta receptor I (TGFßRI).

The TGFß-TGFßR signaling pathway has been reported to play a protective role in the later stages of tumorigenesis via increasing immunosuppressive Treg cells and facilitating the epithelial to mesenchymal transition (EMT). Therefore, inhibition of TGFßR has the potential to enhance antitumor immunity. Herein we disclose the identification and optimization of novel heterobicyclic inhibitors of TGFßRI that demonstrate potent inhibition of SMAD phosphorylation. Application of structure-based drug design to the novel pyrrolotriazine chemotype resulted in improved binding affinity (Ki apparent = 0.14 nM), long residence time (T1/2 > 120 min) and significantly improved potency in the PSMAD cellular assay (IC50 = 24 nM). Several analogs inhibited phosphorylation of SMAD both in vitro and in vivo. Additionally, inhibition of TGFß-stimulated phospho-SMAD was observed in primary human T cells.

Constitutively active receptor tyrosine kinases as oncogenes in preclinical models for cancer therapeutics.

Receptor tyrosine kinases (RTK) remain an area of therapeutic interest because of their role in epithelial tumors, and experimental models specific to these targets are highly desirable. Chimeric receptors were prepared by in-frame fusion of the CD8 extracellular sequence with the cytoplasmic sequences of RTKs. A CD8HER2 fusion protein was shown to form disulfide-mediated homodimers and to transform fibroblasts and epithelial cells. CD8RTK fusion proteins transform rat kidney epithelial cells and impart phenotypes that may reflect signaling specificity inherent in the native receptors. Transgenic expression of CD8HER2 and CD8Met in mice resulted in the formation of salivary and mammary gland tumors. The transgenic tumors allow the derivation of allograft tumors and cell lines that are sensitive to inhibition by small molecule kinase inhibitors. This approach provides excellent cell and tumor models for the characterization of signaling properties of diverse RTKs and for the evaluation of rationally designed antagonists targeting these kinases.

Tumor development by transgenic expression of a constitutively active insulin-like growth factor I receptor.

The insulin-like growth factor I receptor (IGF-IR) is a transmembrane tyrosine kinase that is essential to growth and development and also thought to provide a survival signal for the maintenance of the transformed phenotype. There has been increasing interest in further understanding the role of IGF-I signaling in cancer and in developing receptor antagonists for therapeutic application. We describe herein a novel animal model that involves transgenic expression of a fusion receptor that is constitutively activated by homodimerization. Transgenic mice that expressed the activated receptor showed aberrant development of the mammary glands and developed salivary and mammary adenocarcinomas as early as 8 weeks of age. Xenograft tumors and a cell line were derived from the transgenic animals and are sensitive to inhibition by a novel small-molecule inhibitor of the IGF-IR kinase. This new model should provide new opportunities for further understanding how aberrant IGF-IR signaling leads to tumorigenesis and for optimizing novel antagonists of the receptor kinase.

Cyclooxygenase-2 overexpression in the skin of transgenic mice results in suppression of tumor development.

Significant evidence has accumulated suggesting that the inducible form of cyclooxygenase (COX-2), a central enzyme in the prostaglandin biosynthetic pathway, plays an important role in tumor development. To better understand the role of COX-2 in tumorigenesis, we generated transgenic mice that overexpress COX-2 under control of the human keratin 14 promoter, which allows for expression in the epidermis and some other epithelia. Transgenic mice, referred to as K14.COX2 mice, were readily distinguished from their nontransgenic littermates by the appearance of significant alopecia. Administration of a specific COX-2 inhibitor restored hair growth, indicating that the alopecia was attributable to elevated COX-2 enzymatic activity. Unexpectedly, COX-2 overexpression was found to protect, rather than sensitize, K14.COX2 mice to skin tumor development induced by an initiation/promotion protocol. K14.COX2 transgenics developed tumors at a much lower frequency than did their littermate controls (3.3% versus 93%, respectively, on a FVB background and approximately 25% versus 100%, respectively, on an ICR background) and presented with significantly reduced tumor burdens (average, 0.03 versus 12.7 tumors/mouse, respectively, on a FVB background and 0.5 versus 7.1 tumors/mouse, respectively, on an ICR background). Mice fed a COX-2 inhibitor in utero and as weanlings up to the time of promotion also showed a significant resistance to tumor development. These results clearly raise questions regarding the role of COX-2 and elevated prostaglandin levels in skin tumor development.