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Patients with BRAF-mutant melanoma show significant responses to combined BRAF and MEK inhibition, but most relapse within 2 years. A major reservoir for drug resistance is minimal residual disease (MRD), comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600Eextinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis after BrafV600Eextinction revealed that after an initial surge of immune activation, tumors later became immunologically “cold” after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines that may be central players in the process, being strongly upregulated initially and steeply decreasing as the immune response faded. Therefore, we hypothesized that sustaining the chemokine signaling could impair MRD maintenance through increased recruitment of effector T-cells. We show that intratumoral administration of recombinant Cxcl9, either naked or loaded in microparticles, significantly impaired MRD relapse in BRAF-inhibited tumors, including several complete responses after microparticle-delivered rCxcl9 combined with BRAF and MEK-inhibition. Our experiments constitute a proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of frontline treatment methods.

Supplementary Tables S1-S3

Supplementary Figures S1-S5

Supplementary Figures S6-S10

Patients with BRAF-mutant melanoma show substantial responses to combined BRAF and MEK inhibition, but most relapse within 2 years. A major reservoir for drug resistance is minimal residual disease (MRD), comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600E extinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis after BrafV600E extinction revealed that after an initial surge of immune activation, tumors later became immunologically “cold” after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines as strongly upregulated initially and steeply decreasing as the immune response faded. Therefore, we hypothesized that sustaining chemokine signaling could impair MRD maintenance through increased recruitment of effector T cells. We found that intratumoral administration of recombinant Cxcl9 (rCxcl9), either naked or loaded in microparticles, significantly impaired MRD relapse in BRAF-inhibited tumors, including several complete pathologic responses after microparticle-delivered rCxcl9 combined with BRAF and MEK inhibition. Our experiments constitute proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of first-line treatment methods.

The supplemental figures provide supporting data including controls, synergism studies, dose response curves, validation of knockdown, etc.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis.Significance:Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

Tables s1-9, 11, 13-15. These tables provide statistical data, gene list, and results from TRAP and GSEA that support the conclusions of the manuscript and provide full disclosure of our results.

Supplementary Figures S1-S5 supporting Main Figures 1-5.

Supplementary Figure 1. Co-immuoprecipitation of RhoGDI2 with RhoC in non-bladder cell types Endogenous RhoGDI2 from MDA-MB-231 breast cancer cells (Left), Jurkat lymphocyte cells (Center), or OVCAR-4 ovarian cancer cells (Right) was immunoprecipitated and analyzed by western blot for co-immunoprecipitation of Rac1 and RhoC. Representative western blots of 3 independent experiments shown.

Supplementary Table 2. Differential expression of RhoC/RhoGDI2 signature genes

Fig. S1. Projecting the individual TERTp mutations transcriptional signatures on top of the Onco-GPS map of cellular states (Kim et al. 2017). Fig.S2. Association between the different mRNA signatures analyzed in this study. Fig S3. Expression of epithelial and mesenchymal markers in TERTp mutants (TPM) and wild-type (wt) cancer cell lines. Fig S4. CDH1 expression levels in CCLE lines. Fig S5. GABPa and GABPb1 expression by tissue type. Fig S6. Stratification of TERTp mutant cell lines in The Cancer Therapeutics Response Portal (CTRP) by BRAF status reveals inhibition of growth in the absence of BRAF mutation. Fig. S7. Representative immunoblots of TERT promoter cell lines treated with trametinib for 24 hours.

Table S10 shows IC50 and statistical data from several drugs tested that are presented in figure 1

Supplementary Tables S1-S6 describing patient characteristics, gene lists, LSC signatures, and reagents.

Supplementary Figure and Table Legends. Legends for 3 supplemental figures and 2 tables.

Table S1. TERTp mutations included in this study

Supplementary Figure 3. Depletion of RhoC with a second independent shRNA A. Doubling time for T24T cells expressing shCont or shRhoC-2 was determined by counting cells at 24 h intervals for 96 h. Bargraph shows the mean doubling time{plus minus}s.e. (n=3). *P

The combined loss of CHD1 and MAP3K7 promotes aggressive prostate cancer by unknown mechanisms. Because both of these genes are lost genetically in prostate cancer, they cannot be directly targeted. We applied an established computational systems pharmacology approach (TRAP) to identify altered signaling pathways and associated druggable targets. We compared gene expression profiles of prostate cancer with coloss of CHD1 and MAP3K7 with prostate cancer diploid for these genes using The Cancer Genome Atlas patient samples. This analysis prioritized druggable target genes that included CDK1 and CDK2. We validated that inhibitors of these druggable target genes, including the CDK1/CDK2 inhibitor dinaciclib, had antiproliferative and cytotoxic effects selectively on mouse prostate cells with knockdown of Chd1 and Map3k7. Dinaciclib had stronger effects on prostate cells with suppression of Map3k7 independent of Chd1 and also compared with cells without loss of Map3k7. Dinaciclib treatment reduced expression of homologous recombination (HR) repair genes such as ATM, ATR, BRCA2, and RAD51, blocked BRCA1 phosphorylation, reduced RAD51 foci formation, and increased γH2AX foci selectively in prostate cells with suppression of Map3k7, thus inhibiting HR repair of chromosomal double-strand breaks. Dinaciclib-induced HR disruption was also observed in human prostate cells with knockdown of MAP3K7. Cotreatment of dinaciclib with DNA-damaging agents or PARP inhibitor resulted in a stronger cytotoxic effect on prostate cells with suppression of MAP3K7 compared with those without loss of MAP3K7, or to each single agent.Implications:These findings demonstrate that loss of MAP3K7 is a main contributing factor to drug response through disruption of HR in prostate cancer.

The table shows qRT-PCR data for the knockdown of candidate genes in different bladder cancer cell lines transduced with shRNA containing lentivirus.

Figure legends for all supplemental figures (1-6) describe key findings and experimental details.

Six2 is more highly expressed in the fetal mammary stem cell population compared to adult mouse mammary cells.

The ubiquitin C (UBC) network in bladder cancer.

Detailed Clinical History. Supplemental figure legend Supplemental table legends.

The capacity for tumor cells to metastasize efficiently is directly linked to their ability to colonize secondary sites. Here we identify Six2, a developmental transcription factor, as a critical regulator of a breast cancer stem cell program that enables metastatic colonization. In several triple-negative breast cancer (TNBC) models, Six2 enhanced the expression of genes associated with embryonic stem cell programs. Six2 directly bound the Sox2 Srr2 enhancer, promoting Sox2 expression and downstream expression of Nanog, which are both key pluripotency factors. Regulation of Sox2 by Six2 enhanced cancer stem cell properties and increased metastatic colonization. Six2 and Sox2 expression correlated highly in breast cancers including TNBC, where a Six2 expression signature was predictive of metastatic burden and poor clinical outcome. Our findings demonstrate that a SIX2/SOX2 axis is required for efficient metastatic colonization, underscoring a key role for stemness factors in outgrowth at secondary sites.Significance:These findings provide novel mechanistic insight into stemness and the metastatic outgrowth of triple-negative breast cancer cells.

Legends for supplementary figures

Supplementary materials and methods

List of enriched pathways after GSEA Analysis

Top 18 genes that correlated with IL6 expression in Nanostring dataset (n=12) and TCGA dataset (n=489).

Detailed information (source and working dilution) of antibodies have been used for WB, ICC and IHC.

List of Oligos and Antibodies

Six2 OE leads to a change in the transcriptional program of 4T07 cells and differential expression of stemness-related genes and pathways

Levels of Six2 OE in 4T07 cells is comparable to levels in multiple other human TNBC cell lines with relatively high endogenous Six2 expression.

Purpose:Ovarian cancer has one of the highest deaths to incidence ratios across all cancers. Initial chemotherapy is effective, but most patients develop chemoresistant disease. Mechanisms driving clinical chemo-response or -resistance are not well-understood. However, achieving optimal surgical cytoreduction improves survival, and cytoreduction is improved by neoadjuvant chemotherapy (NACT). NACT offers a window to profile pre- versus post-NACT tumors, which we used to identify chemotherapy-induced changes to the tumor microenvironment.Experimental Design:We obtained matched pre- and post-NACT archival tumor tissues from patients with high-grade serous ovarian cancer (patient, n = 6). We measured mRNA levels of 770 genes (756 genes/14 housekeeping genes, NanoString Technologies), and performed reverse phase protein array (RPPA) on a subset of matched tumors. We examined cytokine levels in pre-NACT ascites samples (n = 39) by ELISAs. A tissue microarray with 128 annotated ovarian tumors expanded the transcriptional, RPPA, and cytokine data by multispectral IHC.Results:The most upregulated gene post-NACT was IL6 (16.79-fold). RPPA data were concordant with mRNA, consistent with elevated immune infiltration. Elevated IL6 in pre-NACT ascites specimens correlated with a shorter time to recurrence. Integrating NanoString (n = 12), RPPA (n = 4), and cytokine (n = 39) studies identified an activated inflammatory signaling network and induced IL6 and IER3 (immediate early response 3) post-NACT, associated with poor chemo-response and time to recurrence.Conclusions:Multiomics profiling of ovarian tumor samples pre- and post-NACT provides unique insight into chemo-induced changes to the tumor microenvironment. We identified a novel IL6/IER3 signaling axis that may drive chemoresistance and disease recurrence.

Nanostring transcriptomic data for all genes include on the PanCancer IO 360 Panel and Reverse Protein Phase Array for GTFB 1064 and 1066 (pre and post-NACT).

Detailed description of methods used for compound 8430 synthesis, cell culture and compound 8430 pharmacokinetics.

In principle, the inhibition of candidate gain-of-function genes defined through genomic analyses of large patient cohorts offers an attractive therapeutic strategy. In this study, we focused on changes in expression of CD24, a well-validated clinical biomarker of poor prognosis and a driver of tumor growth and metastasis, as a benchmark to assess functional relevance. Through this approach, we identified GON4L as a regulator of CD24 from screening a pooled shRNA library of 176 candidate gain-of-function genes. GON4L depletion reduced CD24 expression in human bladder cancer cells and blocked cell proliferation in vitro and tumor xenograft growth in vivo. Mechanistically, GON4L interacted with transcription factor YY1, promoting its association with the androgen receptor to drive CD24 expression and cell growth. In clinical bladder cancer specimens, expression of GON4L, YY1, and CD24 was elevated compared with normal bladder urothelium. This pathway is biologically relevant in other cancer types as well, where CD24 and the androgen receptor are clinically prognostic, given that silencing of GON4L and YY1 suppressed CD24 expression and growth of human lung, prostate, and breast cancer cells. Overall, our results define GON4L as a novel driver of cancer growth, offering new biomarker and therapeutic opportunities. Cancer Res; 76(17); 5175–85. ©2016 AACR.

Histological description of tumors and Cancer antigen 125 (CA125) levels for each patient over time. Units = U/mL

Six2 binding is enriched at the promoters of genes previously shown to be critical for normal and cancer stem cell like function.

Figure S1 - Expression pattern of CD24 in different cell lines used and effect of candidate gene depletion on CD24 protein levels. Figure S2 - siRNA dose-dependent GON4L knockdown and STRING analysis for GON4L interactions. Figure S3 - Effect of YY1 knockdown on GON4L mRNA levels. Figure S4 - Effect of GON4L and YY1 knockdown on tumor growth in vivo and their expression in bladder cancer. Figure S5 - Luciferase reporter assays in control plasmids, endogenous CO-IP for AR and YY1 interaction, and effect of GON4L and YY1 knockdown on AR expression. Figure S6 - AR expression levels in different cancer cell lines, and GON4L and YY1 knockdown as measured by qRT-PCR. Figure S7 - GON4L and YY1 knockdown in AR- cancer cell lines.

Purpose: Genetic analysis of bladder cancer has revealed a number of frequently altered genes, including frequent alterations of the telomerase (TERT) gene promoter, although few altered genes have been functionally evaluated. Our objective is to characterize alterations observed by exome sequencing and sequencing of the TERT promoter, and to examine the functional relevance of histone lysine (K)–specific demethylase 6A (KDM6A/UTX), a frequently mutated histone demethylase, in bladder cancer.Experimental Design: We analyzed bladder cancer samples from 54 U.S. patients by exome and targeted sequencing and confirmed somatic variants using normal tissue from the same patient. We examined the biologic function of KDM6A using in vivo and in vitro assays.Results: We observed frequent somatic alterations in BRCA1 associated protein-1 (BAP1) in 15% of tumors, including deleterious alterations to the deubiquitinase active site and the nuclear localization signal. BAP1 mutations contribute to a high frequency of tumors with breast cancer (BRCA) DNA repair pathway alterations and were significantly associated with papillary histologic features in tumors. BAP1 and KDM6A mutations significantly co-occurred in tumors. Somatic variants altering the TERT promoter were found in 69% of tumors but were not correlated with alterations in other bladder cancer genes. We examined the function of KDM6A, altered in 24% of tumors, and show depletion in human bladder cancer cells, enhanced in vitro proliferation, in vivo tumor growth, and cell migration.Conclusions: This study is the first to identify frequent BAP1 and BRCA pathway alterations in bladder cancer, show TERT promoter alterations are independent of other bladder cancer gene alterations, and show KDM6A loss is a driver of the bladder cancer phenotype. Clin Cancer Res; 20(18); 4935–48. ©2014 AACR.

Bladder cancer network containing debiquitinating enzymes (DUBs), chromosome remodeling genes and genes associated with VEGF signaling.

Six2 regulates Sox2 in multiple human TNBC models.

Supplemental figures (1-6) demonstrate the efficacy of parental SIX1/EYA2 inhibitor, synthesis of compound 8430, endogenous SIX1 and EYA2 expression in tumor lines, as well as 8430's transcriptional impact, pharmacokinetic/toxicity and effect on metastasis.

Normalization factors for each dataset calculated based on housekeeping genes and sample size and power analysis.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFβ signaling and subsequent induction of epithelial–mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC–induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFβ signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer–associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis.Significance:These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.

Six2 and Sox2 mRNA positively correlate in breast cancer patient samples and upregulates genes associated with stemness and poorer prognosis

Supplemental Figure Legends. Legends for Supplemental Figures S1-S8. Also includes Supplemental Reference.

Supplemental Figures S1-S8. SIX2 is overexpressed in breast cancer and correlates with poor prognosis (S1); Six2 KD does not affect proliferation or levels of VEGF-C (S2); Six2 overexpression in 4TO7 cells alters cell morphology but does not increase cell proliferation (S3); Six2 expression in 4TO7 cells increases lung metastasis (S4); Six2 overexpression leads to Zeb2 upregulation in 4TO7 and HMLE cells (S5); SIX2 downregulates miR-8 family members, which when restored to SIX2 overexpressing cells, can reverse the SIX2-induced upregulation of Zeb2 (S6); High levels of SIX2 are found in breast cancer cell lines exhibiting E-cadherin hypermethylation and silencing (S7); SIX2 expression inversely correlates with CDH1 expression in triple negative and non-triple negative breast cancers (S8).

In a substantial fraction of cancers TERT promoter (TERTp) mutations drive expression of the catalytic subunit of telomerase, contributing to their proliferative immortality. We conducted a pan-cancer analysis of cell lines and find a TERTp mutation expression signature dominated by epithelial-to-mesenchymal transition and MAPK signaling. These data indicate that TERTp mutants are likely to generate distinctive tumor microenvironments and intercellular interactions. Analysis of high-throughput screening tests of 546 small molecules on cell line growth indicated that TERTp mutants displayed heightened sensitivity to specific drugs, including RAS pathway inhibitors, and we found that inhibition of MEK1 and 2, key RAS/MAPK pathway effectors, inhibited TERT mRNA expression. Consistent with an enrichment of mesenchymal states in TERTp mutants, cell lines and some patient tumors displayed low expression of the central adherens junction protein E-cadherin, and we provide evidence that its expression in these cells is regulated by MEK1/2. Several mesenchymal transcription factors displayed elevated expression in TERTp mutants including ZEB1 and 2, TWIST1 and 2, and SNAI1. Of note, the developmental transcription factor SNAI2/SLUG was conspicuously elevated in a significant majority of TERTp-mutant cell lines, and knock-down experiments suggest that it promotes TERT expression. Implications: Cancers harboring TERT promoter mutations are often more lethal, but the basis for this higher mortality remains unknown. Our study identifies that TERTp mutants, as a class, associate with a distinct gene and protein expression signature likely to impact their biological and clinical behavior and provide new directions for investigating treatment approaches for these cancers.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. Significance: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.