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Extended Experimental Methods

Supplemental Figure S1. Integrative Analysis of Functional Genomics and Copy Number Variation in Melanoma Cells and Tissues, Supplemental Figure S2. Elastic Net Derived Biomarker Results for Melanoma Survival Genes and Detection of These Biomarkers in TCGA SKCM, Supplemental Figure S3. The SOX10 Regulatory Network Supporting Cell Autonomous Melanoma Cell Growth and Survival, Supplemental Figure S4. SOX10 Addiction Specifies Sensitivity of BRAF Mutant Melanomas to BRAF and MEK Inhibitors In Vitro, Related to Figure 1, Supplemental Figure S5. SOX10 Addiction Specifies Sensitivity of BRAF Mutant Melanomas to BRAF and MEK Inhibitors In Patients, Related to Figure 2, Supplemental Figure S6. Bicluster of melanoma cell lines and chemical compounds in McDermott/Benes GDSC dataset, Related to Figure 3, Supplemental Figure S7. Nomination of TBK1 as a Therapeutic Target for Drug-Resistant Melanoma, Related to Figure 3, Supplemental Figure S8. TBK1/IKKε-Addiction is Conserved In Vivo, Related to Figure 4, Supplemental Figure S9. TBK1/IKKε-Addiction Corresponds to a Cell Autonomous Innate Immune Melanoma Subtype, Related to Figure 5, Supplemental Figure S10. TBK1/IKKε Activate AKT and YAP to Support Survival of the Cell-autonomous Immune Melanoma Subtype, Related to Figure 6, Supplemental Figure S11. 13C glucose and 13C glutamine metabolism at 30m and 2h, Related to Figure 7 and Supplemental Fig. S7, Supplemental Figure S12. Distinct Epigenetic Cell Fate Programs Specify TBK1/IKKε Addiction, Related to Figure 7.

Inhibition of mTOR signaling using the rapalog everolimus is an FDA-approved targeted therapy for patients with lung and gastroenteropancreatic neuroendocrine tumors (NET). However, patients eventually progress on treatment, highlighting the need for additional therapies. We focused on pancreatic NETs (pNET) and reasoned that treatment of these tumors upon progression on rapalog therapy, with an mTOR kinase inhibitor (mTORKi), such as CC-223, could overcome a number of resistance mechanisms in tumors and delay cardiac carcinoid disease. We performed preclinical studies using human pNET cells in vitro and injected them subcutaneously or orthotopically to determine tumor progression and cardiac function in mice treated with either rapamycin alone or switched to CC-223 upon progression. Detailed signaling and RNA sequencing analyses were performed on tumors that were sensitive or progressed on mTOR treatment. Approximately 57% of mice bearing pNET tumors that progressed on rapalog therapy showed a significant decrease in tumor volume upon a switch to CC-223. Moreover, mice treated with an mTORKi exhibited decreased cardiac dilation and thickening of heart valves than those treated with placebo or rapamycin alone. In conclusion, in the majority of pNETs that progress on rapalogs, it is possible to reduce disease progression using an mTORKi, such as CC-223. Moreover, CC-223 had an additional transient cardiac benefit on valvular fibrosis compared with placebo- or rapalog-treated mice. These results provide the preclinical rationale to further develop mTORKi clinically upon progression on rapalog therapy and to further test their long-term cardioprotective benefit in those NET patients prone to carcinoid syndrome. Mol Cancer Ther; 16(11); 2432–41. ©2017 AACR.

Table S1. CC-223-treated mice tend to develop less histopathologic lesions of the heart valves than either rapamycin- or placebo-treated mice. Figure S1. MLN0128 inhibited mTOR signaling in human pNET cell lines in a dose-dependent manner. Figure S2. Mean tumor volumes of rapamycin, CC-223 responders and non-responders over time. Figure S3. Tumor burden, DDC and/or serum serotonin levels in nude mice either orthotopically or subcutaneously injected with BON-luci cells and treated with placebo, rapamycin or CC-223.

Chick Chorioallantoic Membrane (CAM) assay for UM-SCC1 cells.

Cell attachment and migration assays for UM-SCC1 cells.

DNA-PK and Rac1 activity promote UM-SCC47 cell invasion.

RNA sequencing data. Gene ontologies and the associated genes based on biological processes with the most significance based on p-values and corrected q-values.

Purpose: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells.Experimental Design: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors.Results: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity.Conclusions: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes. Clin Cancer Res; 22(8); 2062–73. ©2015 AACR.

Generation and characterization of FA-deficient (HPV positive and HPV negative) HNSCC cell models.

Legends

Supplementary Figures S1-S12. Figure S1. Ingenuity Pathway Analysis (IPA) and Netwalker analysis of the PLX4720-synthetic lethal genes. Figure S2. Effect of Selumetinib on the mitochondrial gene expression in high OxPhos MEL624 cell lines. Figure S3. Differential energy consumption and release in selumetinib-sensitive and resistant melanoma cell lines. Figure S4. Oxygen consumption in MEKi+mTORC1/2i combination-resistant and â??sensitive melanoma cell lines. Figure S5. Cellular effects of FDA approved single agent MEK and BRAF inhibitors Trametinib and Dabrafenib and their combinations with AZD8055. Figure S6. RPPA proteomic analysis of protein lysates from three combination-resistant (1) and three â??sensitive (2) BRAF mutant melanoma cell lines following MEK or mTORC1/2 inhibition. Figure S7. Correlation of MITF expression with selumetinib and AZD8055 combination synergy in 14 melanoma cell lines. Figure S8. Effect of selumetinib and AZD8055 on the cellular localization of MITF and consequence of MITF knockdown Figure S9. Comparative effects of AZD8055 and AZD2014. Figure S10. Drug concentration against percentage of cell growth inhibition. Figure S11. The proliferation of the A375, its resistant clones â??R1 and -R2, and WM35 and its resistant clones in presence of increasing concentrations of selumetinib, AZD8055 and their combination after 72 h treatment was determined as described in Figure S8. Figure S12. A) Ratio of PGC1α and MITF gene expression at the time of disease progression versus pre-treatment in the MGH cohort of patients treated with single-agent vemurafenib (MGH-25) or with dabrafenib + trametinib (all others). B) PGC1α expression in 917 cell lines of the Cancer Cell Line Encyclopedia (CCLE) representing 23 cancer types.

Supplementary Methods. This file contains detailed descriptions of some of the methods used for performing the experiments.

Supplementary Tables 1-2, Figures 1-12 from Basal and Treatment-Induced Activation of AKT Mediates Resistance to Cell Death by AZD6244 (ARRY-142886) in Braf-Mutant Human Cutaneous Melanoma Cells

The majority of melanomas show constitutive activation of the RAS-RAF-MAP/ERK kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway. AZD6244 is a selective MEK1/2 inhibitor that markedly reduces tumor P-MAPK levels, but it produces few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the phosphoinositide 3-kinase (PI3K)-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines showed AZD6244-induced activation of AKT. In contrast, sensitive cell lines showed AZD6244 treatment–induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or insulin-like growth factor I receptor blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells and the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients. Cancer Res; 70(21); 8736–47. ©2010 AACR.

Supplementary Tables S1-S5. Table S1. Patients in the MIA/WH cohort treated with BRAF and/or MEK inhibitors. Table S2. Patients in the MGH patient cohort were treated with the BRAF inhibitor vemurafenib or a combination of BRAF and MEK inhibitors (dabrafenib+trametinib). Table S3. The following melanoma cell lines with known BRAF and NRAS mutation status were seeded in 96 well plates overnight and treated with increasing concentrations (dose range 9nM-6.5mM) of selumetinib (SEL), AZD8055 or their combination. Table S4. Mutations detected in the selumetinib-resistant A375 and WM35 cell line clones compared to their parental cell lines. Table S5. Hugo gene symbols, chromosomal location and gene names of the 202 genes represented in the T200 exon sequencing panel.

Supplementary Figures 1-8, Tables 1-5, Methods from Functional Parsing of Driver Mutations in the Colorectal Cancer Genome Reveals Numerous Suppressors of Anchorage-Independent Growth

Cancer immunotherapy with immune checkpoint inhibitors (ICI) has shown durable clinical benefits in patients with immune-hot tumors. However, the majority of cancers are characterized by an immune-cold tumor microenvironment, which are refractory to ICI therapy. Targeting of tumor synthetic vulnerabilities due to genomic alterations is another promising approach that has proven effective in the clinic (e.g. PARP inhibitors in BRCA-mutant tumors). Here, we aimed to identify therapeutic targets that have dual liabilities in tumor cell survival and the evasion of anti-tumor innate immune responses. Applying machine learning approaches to a manually curated compendium of public and proprietary datasets, we identified and validated several genes with a high potential of conferring tumor-intrinsic cell survival and blocking of innate pro-inflammatory responses in immune-cold tumors. The targeting of these genes induced robust cell killing and activation of innate immune response pathways in specific molecularly-defined tumor populations, including immune-cold cells. A series of highly potent small molecule compounds were established against these targets using structure-based design, which shows robust in vitro and in vivo target engagement and favorable cytotoxic and phenotypic profiles across a large panel of cancer cell lines and PDXs. Collectively, this therapeutic program shows significant promise in treating a multitude of solid tumors as a monotherapy and in combination with currently approved immune therapies. Citation Format: Daniel Ciznadija, Gemechis Degaga, Abdul Mondal, Maxwell Hillbert, Cong Chen, Bikash Debnath, Sonam Mehrotra, Brandon Jones, Ralph Rivero, Kakajan Komurov. Targeting tumor synthetic vulnerabilities and immune evasion mechanisms with CO-1002 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1599.

The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short, and overcoming therapeutic resistance has proven difficult. The presence of chemoresistant leukemic stem cells (LSCs), among other mechanisms, has been implicated as a driver in the observed relapse. Antibody-drug conjugates (ADC) are targeted cancer therapeutics that leverage antigen specificity to deliver a potent cytotoxic payload. Recently, ADCs have shown promise in the clinic, but can also present safety and efficacy concerns. Mylotarg, an FDA approved CD33-directed ADC used to treat Acute Myeloid Leukemia, was the first ADC to be approved. The therapeutic is active against most AML tumors but can lead to severe myelotoxicity given the expression of CD33 on normal myeloid progenitor cells. We leveraged a pharmaco-pheno-multiomic analysis of primary AML tumors to uncover a previously unexplored target expressed on both LSCs and AML blasts, but not normal hematopoetic progenitor cells or other normal tissue. The target also showed capacity to rapidly internalize and has activity of well-established oncogenic kinases associated with expression. A novel monoclonal antibody (IgG1) with favorable binding and biophysical characteristics was established against this novel target and conjugated to a highly potent cytotoxic agent, creating a novel ADC. This ADC shows strong binding across AML tumors with effective internalization and highly potent killing of AML tumors. Citation Format: Garima Kaushik, Bandana Vishwakarma, Marina Bell, Kakajan Komurov. CO-1008, an ADC program targeting leukemic progenitor and blast AML cells, but not normal hematopoetic progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1548.

Oncogenic kinase inhibitors show short-lived responses in the clinic due to high rate of acquired resistance. We previously showed that pharmacologically exploiting oncogene-induced proteotoxic stress can be a viable alternative to oncogene-targeted therapy. Here, we performed extensive analyses of the transcriptomic, metabolomic and proteostatic perturbations during the course of treatment of Her2+ breast cancer cells with a Her2 inhibitor covering the drug response, resistance, relapse and drug withdrawal phases. We found that acute Her2 inhibition, in addition to blocking mitogenic signaling, leads to significant decline in the glucose uptake, and shutdown of glycolysis and of global protein synthesis. During prolonged therapy, compensatory overexpression of Her3 allows for the reactivation of mitogenic signaling pathways, but fails to re-engage the glucose uptake and glycolysis, resulting in proteotoxic ER stress, which maintains the protein synthesis block and growth inhibition. Her3-mediated cell proliferation under ER stress during prolonged Her2 inhibition is enabled due to the overexpression of the eIF2 phosphatase GADD34, which uncouples protein synthesis block from the ER stress response to allow for active cell growth. We show that this imbalance in the mitogenic and proteostatic signaling created during the acquired resistance to anti-Her2 therapy imposes a specific vulnerability to the inhibition of the endoplasmic reticulum quality control machinery. The latter is more pronounced in the drug withdrawal phase, where the de-inhibition of Her2 creates an acute surge in the downstream signaling pathways and exacerbates the proteostatic imbalance. Therefore, the acquired resistance mechanisms to oncogenic kinase inhibitors may create secondary vulnerabilities that could be exploited in the clinic.

Genetic and epigenetic mechanisms determine SCD inhibitor sensitivity and acquired resistance through FOSB in cancer., The lipogenic enzyme stearoyl CoA desaturase (SCD) plays a key role in tumor lipid metabolism and membrane architecture. SCD is often up-regulated and a therapeutic target in cancer. Here, we report the unexpected finding that median expression of SCD is low in glioblastoma relative to normal brain due to hypermethylation and unintentional monoallelic co-deletion with phosphatase and tensin homolog (PTEN) in a subset of patients. Cell lines from this subset expressed undetectable SCD, yet retained residual SCD enzymatic activity. Unexpectedly, these lines evolved to survive independent of SCD through unknown mechanisms. Cell lines that escaped such genetic and epigenetic alterations expressed higher levels of SCD and were highly dependent on SCD for survival. Last, we identify that SCD-dependent lines acquire resistance through a previously unknown FBJ murine osteosarcoma viral oncogene homolog B (FOSB)–mediated mechanism. Accordingly, FOSB inhibition blunted acquired resistance and extended survival of tumor-bearing mice treated with SCD inhibitor.

The nature and role of global transcriptional deregulations in cancers are not fully understood. We report that a large proportion of cancers have widespread defects in mRNA transcription elongation (TE). Cancers with TE defects (TEdeff) display spurious transcription and defective mRNA processing of genes characterized by long genomic length, poised promoters and inducible expression. Signaling pathways regulated by such genes, such as pro-inflammatory response pathways, are consistently suppressed in TEdeff tumors. Remarkably, TEdeff correlates with the poor response and outcome in immunotherapy, but not chemo- or targeted therapy, -treated renal cell carcinoma and metastatic melanoma patients. Forced pharmacologic or genetic induction of TEdeff in tumor cells impairs pro-inflammatory response signaling, and imposes resistance to the innate and adaptive anti-tumor immune responses and checkpoint inhibitor therapy in vivo. Therefore, defective TE is a previously unknown mechanism of tumor immune resistance, and should be assessed in cancer patients undergoing immunotherapy., Transcription elongation (TE) is a key point of inducible gene expression regulation. Here, the authors report widespread TE defects (TEdeff) in a high proportion of cancers that correlate with poor immunotherapy response, highlighting TE defects as potential routes for immune resistance.

A status-quo in targeted cancer therapy is that out of the thousands of somatic alterations found in a cancer cell, alterations only in driver genes determine therapeutic strategy. Despite unimpressive results of some driver-targeted therapies, and given that the majority of genomic alterations in cancer are not ‘drivers’, but ‘passengers’ / bystander alterations, it remains underappreciated whether targeting built-in vulnerabilities imposed by passenger gene alterations may provide therapeutic value. The tumor suppressor PTEN undergoes widespread functional inactivation including deletion in human cancer. PTEN deletion occurs frequently in GBM, sometimes as part of the 10q loss or chromosome 10 monosomy. We discovered that several genes including Stearoyl Co-A Desaturase, SCD (10q24.31), located 12 MB telomeric to PTEN is frequently co-deleted hemizygously and unintentionally in PTEN-deleted cancers. Strikingly, in a subset of GBM, SCD was also epigenetically silenced. A combination of SCD deletion and methylation resulted in two molecular subgroups – one that expressed SCD, and another that showed little or no detectable SCD. SCD, is an integral membrane protein of the endoplasmic reticulum and converts saturated fatty acids to monounsaturated fatty acids (Oleic and palmitoleic acids) that are critical for membrane fluidity and function, and thus SCD is generally overexpressed in most cancers. We show that SCD expressing lines are highly sensitive to multiple SCD inhibitors, while non-expressors are resistant. Despite modest BBB penetration, one SCD inhibitor was remarkably efficient in blocking intracranial tumor growth. SCD is an oxygen-dependent enzyme. We show that SCD retains significant enzymatic activity even in highly hypoxic conditions. Finally, through RNAseq, functional proteomics and ATACseq, we demonstrate an evolutionarily conserved mechanism of acquired resistance to SCD inhibitor through drug-induced acute phase signaling response in multiple SCD expressing cancers.

Stress is integral to tumor evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activate the bioenergetic sensor AMP kinase (AMPK), and tumor cells hijack an AMPK-regulated stress response pathway conserved in normal cells, to survive. Analysis of The Cancer Genome Atlas (TCGA) data revealed that AMPK isoforms are highly expressed in the lethal human cancer Glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumors. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with the cAMP response element binding protein-1 (CREB1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumor bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK supporting the utility of AMPK pharmacological inhibitors in the treatment of GBM.

Inhibition of mTOR signaling using the rapalog everolimus is an FDA-approved targeted therapy for patients with lung and gastroenteropancreatic neuroendocrine tumors (NET). However, patients eventually progress on treatment, highlighting the need for additional therapies. We focused on pancreatic NETs (pNET) and reasoned that treatment of these tumors upon progression on rapalog therapy, with an mTOR kinase inhibitor (mTORKi), such as CC-223, could overcome a number of resistance mechanisms in tumors and delay cardiac carcinoid disease. We performed preclinical studies using human pNET cells in vitro and injected them subcutaneously or orthotopically to determine tumor progression and cardiac function in mice treated with either rapamycin alone or switched to CC-223 upon progression. Detailed signaling and RNA sequencing analyses were performed on tumors that were sensitive or progressed on mTOR treatment. Approximately 57% of mice bearing pNET tumors that progressed on rapalog therapy showed a significant decrease in tumor volume upon a switch to CC-223. Moreover, mice treated with an mTORKi exhibited decreased cardiac dilation and thickening of heart valves than those treated with placebo or rapamycin alone. In conclusion, in the majority of pNETs that progress on rapalogs, it is possible to reduce disease progression using an mTORKi, such as CC-223. Moreover, CC-223 had an additional transient cardiac benefit on valvular fibrosis compared with placebo- or rapalog-treated mice. These results provide the preclinical rationale to further develop mTORKi clinically upon progression on rapalog therapy and to further test their long-term cardioprotective benefit in those NET patients prone to carcinoid syndrome. Mol Cancer Ther; 16(11); 2432–41. ©2017 AACR.

// Eric A. Smith 1, * , Bhavna Kumar 2, 3, * , Kakajan Komurov 1 , Stephen M. Smith 4 , Nicole V. Brown 5 , Songzhu Zhao 5 , Pawan Kumar 2, 3 , Theodoros N. Teknos 2, 3 , Susanne I. Wells 1 1 Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA 2 Department of Otolaryngology–Head and Neck Surgery, The Ohio State University, Columbus, OH, 43210, USA 3 The Ohio State University, Comprehensive Cancer Center, Columbus, OH 43210, USA 4 Department of Pathology, The Ohio State University, Columbus, OH, 43210, USA 5 Center for Biostatistics, The Ohio State University, Columbus, OH, 43210, USA * Co-first author Correspondence to: Susanne I. Wells, email: Susanne.Wells@cchmc.org Theodoros N. Teknos, email: ted.teknos@osumc.edu Keywords: DEK, oropharyngeal squamous cell carcinoma, human papillomavirus, IL6, p16 Received: July 18, 2016 Accepted: February 12, 2017 Published: February 21, 2017 ABSTRACT Oropharyngeal squamous cell carcinomas (OPSCC) are common, have poor outcomes, and comprise two biologically and clinically distinct diseases. While OPSCC that arise from human papillomavirus infections (HPV+) have better overall survival than their HPV- counterparts, the incidence of HPV+ OPSCC is increasing dramatically, affecting younger individuals which are often left with life-long co-morbidities from aggressive treatment. To identify patients which do poorly versus those who might benefit from milder regimens, risk-stratifying biomarkers are now needed within this population. One potential marker is the DEK oncoprotein, whose transcriptional upregulation in most malignancies is associated with chemotherapy resistance, advanced tumor stage, and worse outcomes. Herein, a retrospective case study was performed on DEK protein expression in therapy-naive surgical resections from 194 OPSCC patients. We found that DEK was associated with advanced tumor stage, increased hazard of death, and interleukin IL6 expression in HPV16+ disease. Surprisingly, DEK levels in HPV16- OPSCC were not associated with advanced tumor stage or increased hazard of death. Overall, these findings mark HPV16- OPSCC as an exceptional malignancy were DEK expression does not correlate with outcome, and support the potential prognostic utility of DEK to identify aggressive HPV16+ disease.

Both membrane-proximal and truncation mutations in CSF3R have recently been reported to drive the onset of chronic neutrophilic leukemia (CNL). Here we show that although truncation mutation alone can not induce leukemia, both proximal and compound mutations (proximal and truncation mutations on same allele) are leukemogenic with a disease latency of 90 and 23 days, respectively. Comparative whole-genome expression profiling and biochemical experiments revealed that induced expression of Mapk adaptor protein Ksr1 and enhanced Mapk signaling are crucial to leukemogenesis by CSF3R proximal and compound mutants. Moreover, inhibition of Mek1/2 by trametinib alone is sufficient to suppress leukemia induced by both CSF3R proximal and ruxolitinib-resistant compound mutations. Together, these findings elucidate a Mapk-dependent mechanism of CSF3R-induced pathogenesis, and they establish the rationale for clinical evaluation of MEK1/2 inhibition in CNL.

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.

International audience; Anti-tumor immune responses impede tumor formation, and cancers have evolved many mechanisms of immune evasion. Confirming earlier findings, we show that human tumors with high chromosomal instability (CIN+) are significantly less immunogenic, as judged by tumor lymphocyte infiltration, compared to those with more stable genomes (CIN-). This finding is paradoxical, as genomic instability is expected to evoke an innate immune response. Importantly, CIN+ tumors and cell lines exhibited suppressed expression of proteins involved in MHC class I antigen presentation at least partly due to DNA hypermethylation of the corresponding genes. Using a mouse model of the in vivo evolution of aneuploid tumors, we found that the induction of chromosomal instability in tumor cells is highly immunogenic due to the activation of the STING/TBK1 pathway and consequent increased interferon signaling and antigen presentation. However, tumors evolving under immune pressure suppress the STING/TBK1 and antigen presentation pathways and evade anti-tumor immune responses. In contrast, CIN+ tumors that develop under low immune pressure in both humans and mice retain efficient MHC class I antigen presentation and immunogenicity. Altogether, this study identifies an important mechanism of immune evasion in chromosomally unstable tumors.

Squamous cell carcinoma (SCC) is a global public health burden originating in epidermal stem and progenitor cells (ESPCs) of the skin and mucosa. To understand how genetic risk factors contribute to SCC, studies of ESPC biology are imperative. Children with Fanconi anemia (FA) are a paradigm for extreme SCC susceptibility caused by germline loss-of-function mutations in FA DNA repair pathway genes. To discover epidermal vulnerabilities, patient-derived pluripotent stem cells (PSCs) conditional for the FA pathway were differentiated into ESPCs and PSC-derived epidermal organotypic rafts (PSC-EORs). FA PSC-EORs harbored diminished cell-cell junctions and increased proliferation in the basal cell compartment. Furthermore, desmosome and hemidesmosome defects were identified in the skin of FA patients, and these translated into accelerated blistering following mechanically induced stress. Together, we demonstrate that a critical DNA repair pathway maintains the structure and function of human skin and provide 3D epidermal models wherein SCC prevention can now be explored.

// Vakul Mohanty 1 , Ogulsheker Akmamedova 2 and Kakajan Komurov 3 1 Systems Biology and Physiology Graduate Program, University of Cincinnati, OH, USA 2 Graduate program in Mathematics, Fatih University, Istanbul, Turkey 3 Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, OH, USA Correspondence to: Kakajan Komurov, email: Kakajan.Komurov@cchmc.org Keywords: TCGA, computational cancer biology Received: April 11, 2016 Accepted: June 06, 2016 Published: July 08, 2016 ABSTRACT Chromosomal instability is a hallmark of human cancers, and is characterized by large structural variations in the genome. Such large structural variations are expected to create intrinsic collateral stress due to gene dosage changes in many genes that are co-deleted or co-amplified in large chromosomal segments ( onco-passenger genes). We show that the tumor-toxic effects of gene dosage changes of onco-passenger genes are compensated by the uncoupling of their copy number variations from their expression by means of selective DNA methylation. For example, collateral co-amplification of genes in tumor suppressor pathways, such as the TGF-β and inflammatory signaling pathways, are compensated by DNA hypermethylation to suppress their overexpression, while collateral deletion of pro-oncogenic genes are compensated by DNA hypomethylation to promote their expression from the single remaining allele. Our work reveals an important tumorigenic mechanism of regulation of toxic gene copy number imbalance in tumor cells arising from chromosomal instability, and suggests that targeting the DNA methylation machinery may prevent compensatory regulation of onco-passenger gene expression in chromosomally unstable cancers, and re-activate dormant tumor suppressor pathways for effective therapy.

Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.

ERBB2/HER2-positive breast cancers are associated with poor prognosis; and resistance to HER2-targeted therapy is one of the significant clinical challenges. Trastuzumab and lapatinib are two agents that target the HER2 protein in the clinic and constitute the standard of care for HER2+ breast cancers (BCs). However, the clinical benefits of HER2-targeted therapies are limited due to significant de novo (>60%) and acquired resistance (~100%) to these agents during prolonged phases of treatment. No treatment options exist for metastatic HER2+ BCs that have progressed on HER2-targeted therapy. Therefore, targeting of non-oncogenic vulnerabilities is a promising therapeutic approach in tumors that are resistant to oncogene-targeted therapy. Using an evolutionary selection criterion, we computationally interrogated targetable non-oncogenic vulnerabilities in ERBB2/HER2+ breast cancers, which suffer from a high rate of de novo and acquired resistance to HER2-targeted therapy. We found that the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway is induced in HER2+ breast cancers, partly due to adaptive genomic rearrangements, to counter ER stress caused by increased proteotoxic burden due to heightened HER2/mTOR activity. Genetic and pharmacologic targeting of ERAD lead to irrecoverable ER stress and selectively killed HER2+ breast cancer cells, including those that are resistant to conventional therapy. Cell death due to ERAD inhibition was executed by the IRE1α/JNK pathway, which was suppressed in HER2+ breast cancers due to the activation of JNK phosphatases. Accordingly, inhibiting ERAD in combination with JNK phosphatases synergized in killing HER2+, but not HER2-, breast cancer cells. Our study supports exploiting oncogenic proteotoxicity as a novel therapeutic approach for therapy-refractory breast cancers, and presents an integrated strategy to identify non-oncogenic vulnerabilities in cancers. Citation Format: Navneet Singh, Rashika Joshi, Kakajan Komurov. Integrative systems analyses reveal addiction of HER2+ breast cancers to ERAD for survival. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-53.

Increasing evidence suggests that the tumorigenic process is driven by various forms of transcriptional and epigenetic defects beyond somatic mutations. We recently described a novel non-genomic phenomenon associated with tumorigenesis that we call Transcription Elongation Defects (TEdeff), which is characterized by high levels of truncated transcripts, intron retention, and loss of gene body exon expression. The TEdeff phenotype exists in more than 20% of all human tumors and is linked to immunotherapy resistance (Modur et al, 2018). The aim of this study was to find a new therapeutic strategy for these types of tumors by targeting cellular pathways that are essential for TEdeff tumor survival. Computational analyses of RNA-sequencing data indicate that TEdeff tumors have extensive overexpression of the cytoplasmic protein homeostasis machinery, including several components of autophagy and proteasome degradation pathways. This strongly suggests that these cells may have increased dependency on cellular protein quality control due to elevated proteotoxic stress. Accordingly, we experimentally show that TEdeff cancer cell lines have a profound accumulation of protein aggregates (aggresomes) that appear to be associated with autophagy for clearance. Importantly, TEdeff cells, but not control cells with proficient transcription elongation (TEprof), are highly sensitive to autophagy inhibitors like chloroquine (CQ), but not proteasome inhibitors, which indicates their increased dependency on autophagy for survival. We also show evidence that the proteasomal degradation system may be defective in TEdeff cells, further supporting the hypothesis that these cells are dependent on alternative protein quality control mechanisms. Mechanistically, we propose that blocking the autophagy pathway maximizes proteotoxic stress, due to the accumulation of toxic aggresomes and proteins generated from defective transcripts, leading to a strong synthetic lethality in TEdeff cells. Therefore, autophagy inhibition may be a potent treatment strategy for TEdeff tumors, which are inherently resistant to current immunotherapies. Citation Format: Belal Muhammad, Lisa Vinnedge, KAkajan Komurov. Autophagy inhibition as a new therapeutic strategy for cancers with transcription elongation defects [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 5836.

Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression in vivo, we generated and validated a tetracycline responsive Dek transgenic mouse model referred to as Bi-L-Dek. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing Bi-L-Dek mice to keratin 5 tetracycline transactivator (K5-tTA) mice. Conditional transgene expression was validated in the resulting Bi-L-Dek_K5-tTA mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these data demonstrate that Dek overexpression in the cell of origin for SCC is sufficient to promote esophageal SCC development in vivo., Author summary The DEK oncogene is overexpressed in nearly all human cancers and portends a poor prognosis for many cancer types. High DEK expression causes cancer related phenotypes such as increased cellular proliferation, migration, and invasion in vitro. Despite the well documented link between high DEK expression and cancer, the consequences of Dek overexpression in vivo are poorly understood. To determine if Dek contributes to carcinogenesis in vivo, we generated a Dek inducible transgenic mouse model wherein Dek can be overexpressed in specific tissues and inhibited with the drug doxycycline. We targeted Dek overexpression to keratinocytes, the cell of origin for squamous cell carcinoma, and exposed the mice to the chemical carcinogen 4NQO to induce oral cavity and esophageal carcinogenesis. We found that DEK overexpression was sufficient to increase gross esophageal squamous cell carcinoma incidence and caused a trend toward increased cellular proliferation in adjacent non-tumor tissue. Importantly, we demonstrate for the first time that Dek overexpression specifically targeted to basal keratinocytes is sufficient to promote cellular and squamous cell carcinoma growth in vivo.