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Supplementary Figure 3 from Antitumor Activity of the Hsp90 Inhibitor IPI-504 in HER2-Positive Trastuzumab-Resistant Breast Cancer

Supplementary Figure 1 from Antitumor Activity of the Hsp90 Inhibitor IPI-504 in HER2-Positive Trastuzumab-Resistant Breast Cancer

Supplementary Figure 2 from Antitumor Activity of the Hsp90 Inhibitor IPI-504 in HER2-Positive Trastuzumab-Resistant Breast Cancer

Supplementary Figure Legends 1-3 from Antitumor Activity of the Hsp90 Inhibitor IPI-504 in HER2-Positive Trastuzumab-Resistant Breast Cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is resistant to many available therapies including immunotherapy because of its highly complex tumor microenvironment (TME). PDAC TME consists of a significant proportion of stromal cells, such as endothelial cells, perivascular cells, and cancer-associated fibroblasts (CAFs). Recent work indicates how CAFs can orchestrate the crosstalk cancer and immune cells, and contribute to many aspects of tumor progression, including angiogenesis, senescence, and inflammation. Recent studies based on scRNA-seq have increased understanding of CAF heterogeneity in PDAC in both human and genetically engineered mouse models (GEMMs) is of high interest. To understand the translatability of GEMMs in the setting of PDAC, we conducted a thorough scRNA-seq meta-analysis on CAFs across GEMMs and PDAC human samples. Hereafter, we characterized CAFs multi-dimensionally based on transcriptional, chromatin accessibility, and spatial profiles. Finally, we suggested certain transcription factors may be regulatory drivers of heterogeneous CAF phenotypes in both human and GEMMs.MethodsWe collected publicly available and internally generated scRNA-seq data of PDAC CAFs from human and mouse. After dataset alignment and label transfer, we conducted differential expression analysis across CAF subsets to characterize myofibroblasts (myCAFs) and other CAF subsets of interest. Bioinformatically, we further interrogated CAF heterogeneity in terms of regulatory potential of transcription factors, gene set enrichment, and functional state transition. Complemented by epigenomic assessment, we investigated chromatin accessibility and transcription factor binding availability on the single-cell level. Finally, to investigate the TME organization and spatial neighborhood of cell-to-cell interaction, we explored potential functional differences across location and transcriptional changes of CAF subsets by spatial transcriptomics.ResultsWe found that myofibroblasts (myCAFs) make up a substantial proportion of the CAF population, in both human and mouse TME. In a combination of transcriptional profiling, chromatin accessibility assessment, and spatial transcriptomics, we elucidated potential functional and phenotypic differences within myCAF population and compared to other CAF subsets in the TME. While myofibroblasts are traditionally described as matrix remodeling related, heterogeneity in myofibroblasts may suggest additional roles played by this specific subset. In addition, CAFs in human and mouse share similarities, in terms of transcriptional profiles and phenotypes. The use of GEMMs facilitates our understanding of CAF heterogenous behavior and phenotypes in the PDAC TME.ConclusionsHere, we presented a comprehensive overview of CAF heterogeneity in mouse PDAC models and human datasets. Our observations highlight molecular differences in CAFs, which facilitates our understanding on PDAC stromal microenvironment and translatability in GEMMs in imitating human TME.

KRAS is most frequently mutated in pancreatic adenocarcinomas (PDAC) with more than 90% of cases having a KRAS mutation. This leads to constitutive activation of the downstream signaling pathways, driving tumor cell proliferation whilst co-opting the tumor microenvironment (TME) to promote tumorigenesis and immune escape. The mutant specific KRASG12C inhibitor (AMG510) has been shown to suppress tumor growth and reconfigure the immune TME. However, covalent binding KRASG12C inhibitors have no efficacy on other KRAS alleles, such as G12D and V, the majority of KRAS mutations in PDAC (37% and 28%, respectively). Here, we use a SOS1 inhibitor (BI 1701963) in combination with a MEK inhibitor, blocking two non-redundant steps in the KRAS pathway, on a library of congenic tumor clones derived from an autochthonous mouse model of PDAC deemed the KPCY (Pdx-1-Cre, KRASG12D, p53-/-, YFP+). These tumor lines can generate both “hot” and “cold” TMEs regarding T cell infiltration. We observed that combined SOS1 and MEK inhibition suppressed tumor growth of all KPCY lines irrespective of their cold or hot TME status. In some KPCY lines, CD8 T-cells significantly increased in the TME following combination treatment. We found that the CD8 T-cell increase is restricted to the individual tumor site and resulted from both recruitment and intra-tumoral division. This was accompanied by a decrease of tumor associated macrophages and myeloid-derived suppressor cells. Single-cell RNAseq profiling of the TME was performed on three KPCY lines with distinct immune response to the treatment to characterize cellular composition change induced by the SOS1 and MEK inhibitor combination. To take advantage of the treatment-induced immune TME reconfiguration, we examined combination immune-oncology (IO) targeting T cells and antigen-presenting cells and found that the combination of SOS1i and MEKi with PD-1/CTLA4/CD40 enhanced and prolonged tumor growth inhibition mediated by anti-tumor immunity. Collectively, these results reveal that the combination of SOS1 and MEK inhibitors can be used to convert cold to hot tumors and highlights how we can rationalize combination of cancer-targeted therapy and IO therapy to prolong anti-tumor effects. Citation Format: Robert J Norgard, Pratha Budhani, Sarah O'Brian, Jessica Potts, Brianna Flynn, Xavier Salce, Joshua Tagore, Lucinda Thiede, Joseph Seco, Suzanne Segal, Mark Love, Ania Mikucka, Charlie Cote, Ruby Wasti, Mohanapriya Kamalakannan, Astrid Jeschko, Gabriela Melo-Zainzinger, Angela Mcnabola, Varenka Rodriguez Rodriguez DiBlasi, Joseph Tumang, Ulrike Tontsch-Grunt, Melanie Hinkel, Jeanine Pignatelli, Francesca Trapani, Sandra Martinez-Morilla, Kaja Kostyrko, Marco H Hofmann, Abhishek Kashyap, Emilio Flano, Kang Liu. Combination of SOS1::KRAS inhibitor with a MEK inhibitor reconfigures the immune tumor microenvironment of KRASG12D pancreatic ductal adenocarcinomas and sensitizes to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B23.

Epigenetic modifications confer stable transcriptional patterns in the brain, and both normal and abnormal brain function involve specialized brain regions, yet little is known about brain region-specific epigenetic differences. Here, we compared prefrontal cortex, anterior cingulate gyrus, hippocampus and nucleus accumbens from 6 individuals, performing whole genome bisulfite sequencing for DNA methylation. In addition, we have performed ATAC-seq for chromatin accessibility, and RNA-seq for gene expression in the nucleus accumbens and prefrontal cortex from 6 additional individuals. We found substantial neuron- and brain region-specific differences in both DNA methylation and chromatin accessibility which were largely non-overlapping, and were greatest between nucleus accumbens and the other regions. In contrast, glial methylation and chromatin were relatively homogeneous across brain regions, although neuron/glia ratios varied greatly, demonstrating the necessity for cellular fractionation. Gene expression was also largely the same across glia from different brain regions and substantially different for neurons. Expression was correlated with methylation and accessibility across promoters and known enhancers. Several classes of transcription factor binding sites were enriched at regions of differential methylation and accessibility, including many that respond to synaptic activity. Finally, both regions of differential methylation and those of differential accessibility showed a surprising >10-fold enrichment of explained heritability associated with addictive behavior, as well as schizophrenia- and neuroticism-associated regions, suggesting that common psychiatric illness is mediated through brain region-specific epigenetic marks.

Inhibitors of Bruton’s tyrosine kinase (BTK) possess much promise for the treatment of oncologic and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clinical phenotype of BTK inhibition by systematically examining the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clinical phenotype of chronic BTK inhibition.

Hsp90 facilitates the maturation and stability of numerous oncoproteins, including HER2. The aim of this study was to assess the antitumor activity of the Hsp90 inhibitor IPI-504 in trastuzumab-resistant, HER2-overexpressing breast cancer cells. Therapy with trastuzumab, IPI-504, and the combination of trastuzumab and IPI-504 was evaluated in trastuzumab-sensitive and trastuzumab-resistant cells. Inhibition of protein targets, cell proliferation, and tumor growth was assessed in vitro and in xenograft models. IPI-504 inhibited proliferation of both trastuzumab-sensitive and trastuzumab-resistant cells. Administration of IPI-504 markedly reduced total levels of HER2 and Akt, as well as phosphorylated Akt and mitogen-activated protein kinase (MAPK), to an equal extent in trastuzumab-sensitive and trastuzumab-resistant cells. IPI-504, used as single agent or in combination with trastuzumab, also inhibited in vivo the growth of both trastuzumab-sensitive and -resistant tumor xenografts. As a mechanism for the observed antitumor activity, IPI-504 resulted in a marked decrease in the levels of HER2, Akt, p-Akt, and p-MAPK in trastuzumab-resistant xenografts as early as 12 hours after a single dose of IPI-504. IPI-504–mediated Hsp90 inhibition may represent a novel therapeutic approach in trastuzumab refractory HER2-positive breast cancer. Mol Cancer Ther; 10(5); 817–24. ©2011 AACR.

SummaryUsing a functional approach to investigate the epigenetics of type 2 diabetes (T2D), we combine three lines of evidence—diet-induced epigenetic dysregulation in mouse, epigenetic conservation in humans, and T2D clinical risk evidence—to identify genes implicated in T2D pathogenesis through epigenetic mechanisms related to obesity. Beginning with dietary manipulation of genetically homogeneous mice, we identify differentially DNA-methylated genomic regions. We then replicate these results in adipose samples from lean and obese patients pre- and post-Roux-en-Y gastric bypass, identifying regions where both the location and direction of methylation change are conserved. These regions overlap with 27 genetic T2D risk loci, only one of which was deemed significant by GWAS alone. Functional analysis of genes associated with these regions revealed four genes with roles in insulin resistance, demonstrating the potential general utility of this approach for complementing conventional human genetic studies by integrating cross-species epigenomics and clinical genetic risk.

The Tec family kinase, Bruton’s Tyrosine Kinase (BTK), is one of the key intracellular signaling components proximal to the B cell receptor (BCR). In this study, we utilized an orally-accessible BTK inhibitor (BTKi) to study the functional effects blocking this signaling pathway has on antigen-driven B cell activation in vivo. We report that BTK is required for anti-IgD driven B cell activation in vivo as evidenced by suppression of the surface activation markers CD86 and mRNA transcripts (e.g. c-Myc, Bcl-xL, CCL3, CD98, EBI2, EGR1, EGR2 and IRF4) otherwise rapidly induced upon engagement of the BCR with antigen. Using the T-dependent protein PE and SRBCs as model antigens, we tested for a requirement of BTK in generating antigen-specific IgM and IgG antibody titers and germinal centers, and find that BTK is required for germinal center maintenance. Lastly, we tested for a role of BTK in long-lived PE-specific memory B cell re-activation and report that BTKi significantly suppressed the induction of PE-specific IgM and IgG titers upon antigen re-challenge. The use of BTKi allowed us to bypass the impact a genetically inactivated BTK gene has on B cell development and to assess BTK inactivation in both a wild-type setting and in a temporal manner. These results provide insight into the role of BTK in BCR-driven B cell activation in various antigen naïve and experienced B cell populations.

The two specialized C. elegans distal tip cells (DTCs) provide an in vivo model system for the study of developmentally regulated cell migration. We identified cacn-1/cactin, a well-conserved, novel regulator of cell migration in a genome-wide RNAi screen for regulators of DTC migration. RNAi depletion experiments and analysis of the hypomorphic allele cacn-1(tm3126) indicate that CACN-1 is required during DTC migration for proper pathfinding and for cessation of DTC migration at the end of larval morphogenesis. Strong expression of CACN-1 in the DTCs, and data from cell-specific RNAi depletion experiments, suggest that CACN-1 is required cell-autonomously to control DTC migration. Importantly, genetic interaction data with Rac GTPase activators and effectors suggest that CACN-1 acts specifically to inhibit the mig-2/Rac pathway, and in parallel to ced-10/Rac, to control DTC pathfinding.