Zaw Phyo

Although immunotherapies have been established as a successful treatment option for several types of cancers, additional treatment strategies are necessary to broaden the cohorts of patients who may receive clinical benefit. Common immunological profiling for immunotherapy response depends on assessment of the composition of immune cell types in tissue samples and the periphery. Still, the function and state of each of these cells further contributes to the ultimate clinical benefit of immunotherapeutic response. Additionally, our ability to comprehensively study treatment strategies depends on elucidating the temporal effects different immunotherapy regimens have on the immune system of patients. Single-cell proteomics profiling with CyTOF mass cytometry enables robust protein-level profiling of cell types and is also high-dimensional enough to characterize functional states of immune cells. To construct an immunological framework that may feasibly serve to empower functional assessment of immune cell states for clinical trial monitoring strategies using CyTOF, we are employing single cell trajectory inference methods and non-negative matrix factorization (NMF). We demonstrate this approach using peripheral blood derived CyTOF mass cytometry data to model T cell states at the proteomic level. First, we extract T helper (Th) and T cytotoxic (Tc) cell integrated metrics from CyTOF mass cytometry data obtained from diverse cell marker panel designs, immunological states, and diseases to benchmark that these computational pipelines can accurately reflect Th and Tc activation and exhaustion. Next, we develop a new pipeline that enables the use of continuous functional cell-state metrics from continuous pseudotime and NMF pattern weights as immunological profiling markers from CyTOF that can be applied to cancer immunotherapy clinical trials. For instance, we find CyTOF Tc pseudotime and memory pattern weights are higher in PDAC patients with stable disease treated with ipilimumab and a pancreatic cancer-specific vaccine. Thus, our pipeline has the ability to integrate metrics from mass cytometry data to empower translational analyses for determining patient responses in cancer immunotherapy, such as adaptive immune responses to checkpoint inhibitors and cancer vaccines. Moreover, applying this pipeline to peripheral blood samples obtained before and after treatment also enables us to map further temporal cell state transitions in Tc and Th cells resulting from that treatment. Future work will include leveraging CyTOF T cell integrated metrics in larger scale survival analyses to test the predictive potential in a clinical proof of concept. Citation Format: Dimitrios N. Sidiropoulos, Zaw Phyo, Nicole Gross, Soren Charmsaz, Stephanie Xavier, James Leatherman, Mark Yarchoan, Elizabeth M. Jaffee, Elana J. Fertig, Won Jin Ho. Single cell proteomic quantification of T cell states using mass cytometry for applications in monitoring immune responses in cancer immunotherapy clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1970.

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here, we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8⁺ T cells and macrophage subpopulations toward MHC-II–expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD-1/PD-L1 axis expression in the setting of PTPN22 inhibition could be further leveraged with PD-1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.

New approaches to target MYC include the stabilization of a guanine-rich, G-quadruplex (G4) tertiary DNA structure in the NHE III region of its promoter. Recent screening of a small molecule microarray platform identified a benzofuran, D089, that can stabilize the MYC G4 and inhibit its transcription. D089 induced both dose- and time-dependent multiple myeloma cell death mediated by endoplasmic reticulum induced stress. Unexpectedly, we uncovered two mechanisms of cell death: cellular senescence, as evidenced by increased levels of p16, p21 and γ-H2AX proteins and a caspase 3-independent mechanism consistent with pyroptosis. Cells treated with D089 exhibited high levels of the cleaved form of initiator caspase 8; but failed to show cleavage of executioner caspase 3, a classical apoptotic marker. Cotreatment with the a pan-caspase inhibitor Q-VD-OPh did not affect the cytotoxic effect of D089. In contrast, cleaved caspase 1, an inflammatory caspase downstream of caspases 8/9, was increased by D089 treatment. Cells treated with D089 in addition to either a caspase 1 inhibitor or siRNA-caspase 1 showed increased IC₅₀ values, indicating a contribution of cleaved caspase 1 to cell death. Downstream effects of caspase 1 activation after drug treatment included increases in IL1B, gasdermin D cleavage, and HMGB1 translocation from the nucleus to the cytoplasm. Drug treated cells underwent a ‘ballooning’ morphology characteristic of pyroptosis, rather than ‘blebbing’ typically associated with apoptosis. ASC specks colocalized with NLRP3 in proximity ligation assays after drug treatment, indicating inflammasome activation and further confirming pyroptosis as a contributor to cell death. Thus, the small molecule MYC G4 stabilizer, D089, provides a new tool compound for studying pyroptosis. These studies suggest that inducing both tumor senescence and pyroptosis may have therapeutic potential for cancer treatment.

Background: T cell exhaustion is a hyporesponsive state commonly found in cancer and is characterized by overexpression of inhibitory receptors and secretion of immunosuppressive cytokines. Immune checkpoint inhibitors (ICIs) target inhibitory receptors such as PD-1 and CTLA-4 to reinvigorate exhausted T cells and promote anti-tumor responses. While ICIs have improved clinical outcomes, no biomarker to date reliably determines the extent of T cell exhaustion. Since robust T cell receptor (TCR) signaling is critical for effective antitumor immunity, we hypothesized that interrogating the phospho-signaling states within the T cells along with their functional states will enhance our understanding of T cell exhaustion and yield potential biomarkers. Methods: In this study, we developed a validated mass cytometry panel of 24 markers to quantify phosphorylation states of 8 intracellular kinases involved in the TCR signaling within multiple CD8+ T cell subtypes. To identify potential differences between the local and peripheral immunological responses, we profiled CD8+ T cells derived from four sites (tumor, tumor draining lymph node, spleen and peripheral blood) in mice bearing MC38-induced flank tumors. We performed a clustering analysis of the dataset in aggregate using FlowSOM and visualized the results with UMAP, a dimensionality reduction algorithm, to compare the phospho-profiles of CD8+ T cell subtypes in all four sites. Results: Bulk analysis of CD8+ T cells demonstrates site-specific concordance between phosphoprotein markers and T cell functional markers (i.e. Ki67 and Granzyme B) with the highest expression in tumor followed by tumor draining lymph node, spleen and peripheral blood. Based on the results from the clustering analysis, site-specific breakdown of CD8+ T cells revealed high prevalence of effector, memory, and exhausted memory T cells in tumors compared to other sites where naïve T cells predominated. Despite these variations in CD8+ T cell immunophenotypic distribution, TCR signaling profiles correlated strongly with specific T cell subtypes across all sites. Conclusion: These data demonstrate that subtype-specific TCR signaling is preserved systemically and that phospho-immune subtyping of CD8+ T cells in the peripheral blood may be used to identify T cell exhaustion states that are found in the tumor microenvironment. Further studies will be conducted to investigate the changes in the TCR phospho-profiles in response to ICI treatment and their associated functions. Citation Format: Zaw Phyo, Rohan Verma, Won J. Ho, Elizabeth M. Jaffee. Site-specific profiling of TCR signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4475.

Remarkable progress in cancer immunology has revolutionized cancer therapy. The majority of patients, however, do not respond to immunotherapeutic options, warranting the ongoing search for better strategies. Leveraging the established role of protein tyrosine phosphatase non-receptor type 22 (PTPN22) in autoimmune diseases, we hypothesized that PTPN22 is a novel target for cancer immunotherapy. PTPN22 is a physiologic regulator of T cell receptor (TCR) signaling acting by dephosphorylating activating tyrosine residues in Lck and Zap70. We first confirmed the relevance of PTPN22 expression by exploring its expression in multiple human cancer types using The Cancer Genome Atlas (TCGA). PTPN22 expression positively correlated with T cell and M1 macrophage gene signatures and immune regulatory genes, especially inflamed tumor types. Next, we directly investigated the role of PTPN22 in antitumor immunity by comparing in vivo tumor characteristics in wild-type (WT) and PTPN22 knockout (KO) mice. Consistent with our hypothesis, PTPN22 KO mice resisted MC38 and EG7 tumors significantly compared with WT. Mass cytometry (CyTOF) profiling of the immune tumor microenvironment demonstrated that MC38 tumors in PTPN22 KO mice were infiltrated with greater numbers of T cells, particularly CD8+ T cells expressing granzyme B and PD1. To further delineate the effects of PTPN22 KO on TCR signaling, we established an optimized CyTOF panel of 9 phosphorylation sites involved in the TCR signaling pathway, including two enzymatic substrates of PTPN22 (Lck Y394 and Zap70 Y493) and 15 immune subtyping markers. CyTOF phospho-profiling of CD8 T cells from tumor-bearing mouse spleens and the peripheral blood of immunotherapy-naïve cancer patients showed that the phosphorylated state of Zap70 Y493 correlated strongly with granzyme B expression. Furthermore, phospho-profiling of tumor-infiltrating CD8+ T cells (a measure of T cell activation) revealed the highest TCR-pathway phosphorylation levels in memory CD8+ T cells that express PD1. The difference in phosphorylation levels between WT and PTPN22 KO was most pronounced for Lck Y394. Based on these findings, we then hypothesized that PD1 inhibition will further enhance the antitumor immune responses promoted by the lack of PTPN22. Indeed, PTPN22 KO mice bearing MC38 and EG7 tumors responded more significantly to anti-PD1 therapy when compared with tumor-bearing WT mice. Finally, we treated WT tumor bearing mice with two different small molecule inhibitors of PTPN22, one previously published compound, LTV1, and one novel compound, L1 (discovered through structure based synthesis). While both inhibitors phenocopied the PTPN22 KO mice in resisting MC38 tumor growth, L1 treatment gave an immune profile that resembled what was observed in tumor-bearing PTPN22 KO mice. Taken together, our results demonstrate that PTPN22 is a novel systemic target for augmenting antitumor immunity. Citation Format: Won Jin Ho, Jianping Lin, Ludmila Danilova, Zaw Phyo, Soren Charmsaz, Aditya Mohan, Todd Armstrong, Ben H. Park, Elana J. Fertig, Zhong-Yin Zhang, Elizabeth M. Jaffee. PTPN22 is a systemic target for augmenting antitumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3398.

Cutaneous T cell lymphomas (CTCL) comprise of a heterogeneous group of non-Hodgkin lymphomas derived from skin-homing T cells. Variation in clinical presentation and lack of definitive molecular markers make diagnosis especially challenging. The biology of CTCL remains elusive and clear links between genetic aberrations and epigenetic modifications that would result in clonal T cell expansion have not yet been identified. Nevertheless, in recent years, next generation sequencing (NGS) has enabled a much deeper understanding of the genomic landscape of CTCL by uncovering aberrant genetic pathways and epigenetic dysregulations. Additionally, single cell profiling is rapidly advancing our understanding of patients-specific tumor landscape and its interaction with the surrounding microenvironment. These studies have paved the road for future investigations that will explore the functional relevance of genetic alterations in the progression of disease. The ultimate goal of elucidating the pathogenesis of CTCL is to establish effective therapeutic targets with more durable clinical response and treat relapsing and refractory CTCL.

G-quadruplexes (G4s) are noncanonical DNA structures that frequently occur in the promoter regions of oncogenes, such as MYC, and regulate gene expression. Although G4s are attractive therapeutic targets, ligands capable of discriminating between different G4 structures are rare. Here, we describe DC-34, a small molecule that potently downregulates MYC transcription in cancer cells by a G4-dependent mechanism. Inhibition by DC-34 is significantly greater for MYC than other G4-driven genes. We use chemical, biophysical, biological, and structural studies to demonstrate a molecular rationale for the recognition of the MYC G4. We solve the structure of the MYC G4 in complex with DC-34 by NMR spectroscopy and illustrate specific contacts responsible for affinity and selectivity. Modification of DC-34 reveals features required for G4 affinity, biological activity, and validates the derived NMR structure. This work advances the design of quadruplex-interacting small molecules to control gene expression in therapeutic areas such as cancer.

MYC is deregulated in many malignancies and its aberrant expression is associated with tumorigenesis and tumor maintenance. We employed a therapeutic strategy to target both MYC transcription, and protein stability. Previously, we developed a small molecule microarray and screened it for MYC G4-binding compounds. The stabilization of G-quadraplex (G4) structures by nucleic acid binding small molecules in the NHE III region of the MYC promoter slows MYC transcription. We identified a benzofuran containing small molecule that could selectively and reversibly stabilize MYC-G4 DNA and reduce MYC expression. This compound demonstrated a G4-dependent mechanism of action showing toxicity in multiple myeloma cell lines containing a G4 in their MYC promoter whereas minimal effects were seen in a Burkitt’s lymphoma line lacking the MYC G4 sequence. Gene expression analysis validated that this compound selectively inhibited MYC and other MYC effectors, but did little to limit the expression of other G4 containing genes. The synthesis of more than 15 analogs identified a new benzofuran-containing analog with enhanced inhibitory activity in a diverse panel of human cancer cell lines (NCI-60), including a number of myeloma cell lines. EDGE-seq data confirmed that the new analog is similar to the original compound in having higher affinity for the G4 in MYC compared to the G4 in BCL2, KRAS, VEGFA and HIF1A. In addition, the analog was synergistic with entinostat, an HDAC inhibitor that can affect MYC protein stability, in limiting myeloma cells, but not PBMCs. The more potent growth inhibitory activity of the new analog allowed us to generate sufficient quantities to evaluate its pharmacokinetics and toxicity in mice. The compound was well tolerated in a dose-escalation toxicity study; no adverse effects were observed and preliminary pharmacokinetic studies showed promising serum bioavailability and exposure properties when administered either intravenously, intraperitoneally or orally. In an assessment of short term in vivo activity, MYC protein expression was inhibited in multiple myeloma xenografts. Longer term studies to evaluate the anti-tumor activity of the compound are currently in progress. Thus, our data provide evidence that small molecule stabilization of the MYC G4 can drive transcriptional silencing of oncogenic MYC both in vitro and in vivo. Furthermore, synergistic tumor cell killing was achieved when the MYC G4 inhibitor was coupled with HDAC inhibition. Citation Format: Snehal M. Gaikwad, David R. Calabrese, Elena C. Leon, John K. Simmons, Shuling Zhang, Aleksandra Michalowski, Sayeh Gorjifard, Zaw Phyo, Daniel Connors, John S. Schneekloth, Beverly A. Mock. Effective targeting of MYC expression with a novel nucleic acid binding (G4-quadruplex) small molecule coupled with HDAC inhibition synergizes to limit myeloma growth [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 194. doi:10.1158/1538-7445.AM2017-194