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KRAS and TAK1 dependency in human and mouse colon cancer.

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Recently developed inhibitors of polymerase theta (POLθ) have demonstrated synthetic lethality in BRCA-deficient tumor models. To examine the contribution of the immune microenvironment to antitumor efficacy, we characterized the effects of POLθ inhibition in immunocompetent models of BRCA1-deficient triple-negative breast cancer (TNBC) or BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). We demonstrate that genetic POLQ depletion or pharmacological POLθ inhibition induces both innate and adaptive immune responses in these models. POLθ inhibition resulted in increased micronuclei, cGAS/STING pathway activation, type I interferon gene expression, CD8+ T cell infiltration and activation, local paracrine activation of dendritic cells and upregulation of PD-L1 expression. Depletion of CD8+ T cells compromised the efficacy of POLθ inhibition, whereas antitumor effects were augmented in combination with anti-PD-1 immunotherapy. Collectively, our findings demonstrate that POLθ inhibition induces immune responses in a cGAS/STING-dependent manner and provide a rationale for combining POLθ inhibition with immune checkpoint blockade for the treatment of HR-deficient cancers.

Cancers deficient in homologous recombination (HR) repair secondary to mutations in genes such as BRCA1 or BRCA2, are dependent on alternative DNA damage response (DDR) pathways to maintain genomic integrity, rendering them susceptible to synthetic lethal targeting of these pathways. Recently, inhibitors of polymerase theta (POLθ, encoded by POLQ), the critical enzyme in microhomology-mediated end-joining (MMEJ), have been shown to be synthetic lethal with HR repair deficiency (Zhou et al. Nature Cancer 2021). Both HR and MMEJ require nucleolytic DNA end-resection to allow for DSB repair, and we have previously shown that MMEJ acts as a barrier to DNA end-resection at DSBs (Patterson-Fortin et al. Cancer Research 2022). Given the synthetic lethality between HR and MMEJ leads to unrestrained DNA end-resection generating chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, we hypothesized that POLθ inhibition in HR-deficient cancers would activate the cGAS/STING innate immune response pathway and facilitate immunotherapy. To investigate the interactions of POLθ inhibition with the immune microenvironment in HR-deficient cancers, we used human cell lines and genetically modified mouse models representative of BRCA1-deficient triple-negative breast cancer (TNBC) and BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). Genetic or pharmacological inhibition of POLθ using novobiocin, a first-in-class inhibitor of the POLθ ATPase domain, induced significantly increased cytosolic dsDNA contained in micronuclei. This free DNA was sensed by the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS), increasing synthesis of cGAMP, in HR-deficient tumor cells but not in HR-proficient tumor cells. Increased cGAMP bound to and activated stimulator of interferon genes (STING), triggering phosphorylation of TBK1 and ultimately of IRF3. Activation of the cGAS/STING pathway by POLθ inhibition drove the expression of type I interferon response elements, including PD-L1. Depletion of STING by siRNA or by CRISPR abrogated this pro-inflammatory signaling and abolished the anti-tumor efficacy of novobiocin-mediated POLθ inhibition. Pharmacologic inhibition of POLθ enhanced Granzyme B+ CD8+ T-cell tumor infiltration. Importantly, antibody-mediated depletion of CD8+ T-cell severely compromised the anti-tumor efficacy of novobiocin-mediated POLθ inhibition, whereas anti-tumor activity of POLθ inhibition was augmented with the addition of either anti-PD-1 or anti-CTLA-4 antibodies. These results demonstrate that POLθ inhibition in HR-deficient cancers mediates a pro-inflammatory response in HR-deficient TNBC or PDAC tumor microenvironments, and that immune checkpoint blockade inhibition enhances the therapeutic efficacy of POLθ inhibition. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero, Gerburg M. Wulf, Brian M. Wolpin, Ben Z. Stanger, Andrew J. Aguirre, James M. Cleary, Alan D. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers [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 6190.

Objective To compare cancer detection rate (CDR), patient recall, and interpretation time of a full protocol MRI (fpMRI) to an abbreviated MRI protocol (abMRI) in high-risk women. Methods This retrospective study was approved by the IRB. All sequential high-risk screening MRI examinations performed between January 1, 2013, and December 31, 2016, were included. Breast radiologists reviewed patient history, prior images, and abMRI images and recorded their interpretation. Time for interpretation reflected review of the MRI study but not dictation or report generation. Following a minimum 30-day washout period, radiologists interpreted the fpMRI, with interpretation and timing recorded. Data collected included CDR, interpretation time, and patient recall rate. Statistical analyses utilized were Cohen’s kappa coefficient, Student’s t-test, and McNemar’s test. Results Included were 334 MRI examinations of 286 women. Interpretation time was 60.7 seconds for the abMRI compared to 99.4 seconds for the fpMRI, with an average difference of 38.7 ± 5.4 seconds per patient (P < 0.0001). Recall rates were comparable: the abMRI recall rate was 82/334 (24.6%) and the fpMRI 81/334 (24.3%). All five cancers included were detected by both protocols with equal recall rate. However, there were more recommendations for biopsy with the fpMRI, although this difference was not statistically significant. Conclusion The abMRI demonstrated comparable CDR to fpMRI, with shortened interpretation time and similar recall rates. Implementing an abMRI to screen high-risk women reduces imaging and interpretation time, thereby improving cost-effectiveness and the patient experience without reduction in cancer detection.

Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor–induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8+ T-cell infiltration and activation in vivo, and that CD8+ T-cell depletion severely compromises antitumor efficacy. Olaparib-induced T-cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-deficient compared with HR-proficient TNBC cells and in vivo models. CRISPR-mediated knockout of STING in cancer cells prevents proinflammatory signaling and is sufficient to abolish olaparib-induced T-cell infiltration in vivo. These findings elucidate an additional mechanism of action of PARP inhibitors and provide a rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. Significance: This work demonstrates cross-talk between PARP inhibition and the tumor microenvironment related to STING/TBK1/IRF3 pathway activation in cancer cells that governs CD8+ T-cell recruitment and antitumor efficacy. The data provide insight into the mechanism of action of PARP inhibitors in BRCA-associated breast cancer. This article is highlighted in the In This Issue feature, p. 681

Tissue marker placement after image-guided breast biopsy has become a routine component of clinical practice. Marker placement distinguishes multiple biopsied lesions within the same breast, prevents re-biopsy of benign lesions, enables multi-modality correlation, guides pre-operative localization and helps confirm surgical target removal. Numerous breast tissue markers are currently available, with varied shapes, composition, and associated bio-absorbable components. This review serves to familiarize the breast interventionalist with the tissue markers most widely available in the United States today and to provide guidance regarding selection of appropriate markers for various clinical settings.

Discovery of a new HDAC6 inhibitor reveals role for HDAC6 in the regulation of glycolytic metabolism., Triple-negative breast cancer (TNBC) is a subtype of breast cancer without a targeted form of therapy. Unfortunately, up to 70% of patients with TNBC develop resistance to treatment. A known contributor to chemoresistance is dysfunctional mitochondrial apoptosis signaling. We set up a phenotypic small-molecule screen to reveal vulnerabilities in TNBC cells that were independent of mitochondrial apoptosis. Using a functional genetic approach, we identified that a “hit” compound, BAS-2, had a potentially similar mechanism of action to histone deacetylase inhibitors (HDAC). An in vitro HDAC inhibitor assay confirmed that the compound selectively inhibited HDAC6. Using state-of-the-art acetylome mass spectrometry, we identified glycolytic substrates of HDAC6 in TNBC cells. We confirmed that inhibition or knockout of HDAC6 reduced glycolytic metabolism both in vitro and in vivo. Through a series of unbiased screening approaches, we have identified a previously unidentified role for HDAC6 in regulating glycolytic metabolism.

Background Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Methods Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Results Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors in vivo and is mediated by CD8+ T-cells. Conclusions Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC.

Background: The heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB), however, mouse models to test this are currently lacking. Proper choice and use of pre-clinical models are necessary for rigorous, pre-clinical studies to rapidly move laboratory findings into the clinic to treat patients. Methods: To elucidate how tumor latency and the heterogeneity of the TME contributes to ICB resistance, we performed comprehensive characterization of the TME using quantitative flow-cytometry and RNA expression analysis (NanoString) utilizing three distinct breast cancer models, all derived from the same autochthonous model. Tumor cells were obtained from the commonly used MMTV-PyMT murine breast cancer model and 1E6, 1E5 or 1E4 cells were either immediately injected into the mammary fat pad of FVB/NJ wild type mice or frozen (i.e. the tumor inoculation derived from the MMTV-PyMT tumors were never cultured). We then correlated the immunophenotyping to the efficacy observed from ICB. Results: These studies reveal that the number of cells used to generate syngeneic tumors significantly influences tumor latency, the infiltrating leukocyte population and response to ICB. The 4 models had vastly different TMEs which correlated to responses to ICB. Compared to the autochthonous model, all three syngeneic models had significantly more tumor infiltrating lymphocytes (TILs; CD3 + , CD4 + , and CD8 + ) and higher proportions of PD-L1 positive myeloid cells, whereas the MMTV-PyMT model had the highest frequency of myeloid cells out of total leukocytes. Increased TILs correlated with response to anti-PD-L1 and anti-CTLA-4 therapy; but tumor cell PD-L1expression and T-cell PD-1 expression did not. Conclusions: Here we have identified ICB-sensitive and resistant breast cancer models, generated from the same tumor cell inoculum. These models represent an opportunity to further interrogate the TME for breast cancer treatment and provide novel insights into therapeutic combinations and response to ICB. We believe this work serves as an important resource for the field to inform proper mouse model selection for pre-clinical studies.

BackgroundThe heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB); however, mouse models to test this are currently lacking. Proper selection and use of preclinical models are necessary for rigorous, preclinical studies to rapidly move laboratory findings into the clinic.MethodsThree versions of a common syngeneic model derived from the MMTV-PyMT autochthonous model were generated by inoculating 1E6, 1E5, or 1E4 cells derived from the MMTV-PyMT mouse into wildtype recipient mice. To elucidate how tumor latency and TME heterogeneity contribute to ICB resistance, comprehensive characterization of the TME using quantitative flow-cytometry and RNA expression analysis (NanoString) was performed. Subsequently, response to ICB was tested. These procedures were repeated using the EMT6 breast cancer model.ResultsThe 3 syngeneic versions of the MMTV-PyMT model had vastly different TMEs that correlated to ICB response. The number of cells used to generate syngeneic tumors significantly influenced tumor latency, infiltrating leukocyte populations, and response to ICB. These results were confirmed using the EMT6 breast cancer model. Compared to the MMTV-PyMT autochthonous model, all 3 MMTV-PyMT syngeneic models had significantly more tumor-infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and higher proportions of PD-L1-positive myeloid cells, whereas the MMTV-PyMT autochthonous model had the highest frequency of myeloid cells out of total leukocytes. Increased TILs correlated with response to anti-PD-L1 and anti-CTLA-4 therapy, but PD-L1expression on tumor cells or PD-1 expression of T cells did not.ConclusionsThese studies reveal that tumor cell number correlates with tumor latency, TME, and response to ICB. ICB-sensitive and resistant syngeneic breast cancer models were identified, in which the 1E4 syngeneic model was most resistant to ICB. Given the lack of benefit from ICB in breast cancer, identifying robust murine models presented here provides the opportunity to further interrogate the TME for breast cancer treatment and provide novel insights into therapeutic combinations to overcome ICB resistance.

Screening mammography has been shown to decrease the mortality from breast cancer by as much as 40% when screening women annually beginning at age 40. Since the initial implementation of mammography, breast imaging has evolved to include numerous other imaging modalities, including full-field digital mammography, digital breast tomosynthesis, ultrasound, magnetic resonance imaging, gamma imaging of the breast referred to as both molecular breast imaging and breast-specific gamma imaging, as well as contrast-enhanced spatial mammography. The goal of these technologic advances is to detect small, early-stage breast cancer, thus providing patients with the best chance of cure with the least amount of treatment-associated morbidity. With the increasing use of mammography and the detection of small, indeterminate lesions, the need for minimally invasive approaches for tissue acquisition has also increased to allow for definitive pathologic assessment. However, with the detection of smaller lesions comes the challenge of developing methods for tissue acquisition that are accurate, cost-effective, and safe, particularly because approximately 80% of breast biopsies result in a benign finding.

BACKGROUND amp; AIMS: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termedMallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation. METHODS: Weanalyzed livers of aged Krt18(-/-) mice that spontaneously developed in the majority of cases SH-associated HCC independent of sex. Interestingly, the hepatic lipid profile in Krt18(-/-) mice, which accumulate KRT8, closely resembles human SH lipid profiles and shows that the excess of KRT8 over KRT18 determines the likelihood to develop SH-associated HCC linked with enhanced lipogenesis. RESULTS: Our analysis of the genetic profile of Krt18(-/-) mice with 26 human hepatoma cell lines and with data sets of amp;gt;300 patients with HCC, where Krt18(-/-) gene signatures matched human HCC. Interestingly, a high KRT8/18 ratio is associated with an aggressive HCC phenotype. CONCLUSIONS: We can prove that intermediate filaments and their binding partners are tightly linked to hepatic lipid metabolism and to hepatocarcinogenesis. We suggest KRT8/18 ratio as a novel HCC biomarker for HCC. Funding Agencies|Kurt und Senta Herrmann Stiftung; Austrian Genome Programme GEN-AU; DFG [MA1316-15, MA1316-17, MA1316-19, MA1316-21, INST 268/230-1]; German Research Foundation [STR 1095/4-2]; Else Kroner Exzellenzstipendium; Innovative Medicines Initiative Joint Undertaking from the European Unions Seventh Framework Programme (FP7/2007-2013) [115234]; EFPIA companies

Tissue marker placement after image-guided breast biopsy has become a routine component of clinical practice. Marker placement distinguishes multiple biopsied lesions within the same breast, prevents re-biopsy of benign lesions, enables multi-modality correlation, guides pre-operative localization and helps confirm surgical target removal. Numerous breast tissue markers are currently available, with varied shapes, composition, and associated bio-absorbable components. This review serves to familiarize the breast interventionalist with the tissue markers most widely available in the United States today and to provide guidance regarding selection of appropriate markers for various clinical settings.

The purpose of this study was to evaluate our initial experience with gamma imaging-guided vacuum-assisted breast biopsy in women with abnormal findings.A retrospective review of patients undergoing breast-specific gamma imaging (BSGI), also known as molecular breast imaging (MBI), between April 2011 and October 2015 found 117 nonpalpable mammographically and sonographically occult lesions for which gamma imaging-guided biopsies were recommended. Biopsy was performed with a 9-gauge vacuum-assisted device with subsequent placement of a titanium biopsy site marker. Medical records and pathologic findings were evaluated.Of the 117 biopsies recommended, 104 were successful and 13 were canceled. Of the 104 performed biopsies, 32 (30.8%) had abnormal pathologic findings. Of those 32 biopsies, nine (28.1%) found invasive cancers, six (18.8%) found ductal carcinoma in situ (DCIS), and 17 (53.1%) found high-risk lesions. Of the 17 high-risk lesions, there were three (17.6%) lobular carcinomas in situ, five (29.4%) atypical ductal hyperplasias, two (11.8%) atypical lobular hyperplasias, one (5.9%) flat epithelial atypia, and six (35.3%) papillomas. Two cases of atypical ductal hyperplasia were upgraded to DCIS at surgery. The overall cancer detection rate for gamma imaging-guided biopsy was 16.3%. In this study, gamma imaging-guided biopsy had a positive predictive value of total successful biopsies of 16.3% for cancer and 30.8% for cancer and high-risk lesions.Gamma imaging-guided biopsy is a viable approach to sampling BSGI-MBI-detected lesions without sonographic or mammographic correlate. Our results compare favorably to those reported for MRI-guided biopsy.

Molecular breast imaging (MBI), also called breast-specific gamma imaging (BSGI), has been an integral component of our breast imaging practice for over a decade. Unlike mammography and ultrasound that are based on anatomy, MBI is a physiologic approach to breast cancer detection. MBI detects additional foci of occult breast cancer in 9.0% of women with newly diagnosed breast cancer, has a high sensitivity for detecting high-risk lesions, and detects 98% of invasive breast cancer and 91.0% of ductal carcinoma in situ. Furthermore, in surveillance of high-risk women, BSGI/MBI detects occult cancer in up to 16.5 per 1000 women. This modality is increasingly being used to assess response to treatment in women undergoing neo-adjuvant chemotherapy and for adjunct screening in women with dense breasts. It has been shown to influence surgical management in nearly a quarter of women with newly diagnosed breast cancer. The Society of Nuclear Imaging has established clinical indications and The American College of Radiology has established appropriateness criteria as well as an accreditation program for MBI. A BIRADS-like lexicon for MBI has also been described. Initially, MBI utilized 10-20 mCi of 99 mTc sestamibi, however, recent studies have reported the use of 5-10 mCi with equal sensitivity to the higher dose of radiotracer. There are over 300 studies in the literature about MBI/BSGI with increasing integration of MBI into clinical practice. This chapter will describe the history, current literature and indications, clinical use, approach to biopsy and integration of MBI into clinical practice.

Magnetic resonance imaging (MRI) is the most sensitive exam for detecting breast cancer. The American College of Radiology recommends women with 20% or greater lifetime risk of developing breast cancer be screened annually with MRI. However, other high-risk populations would also benefit. Hartmann et al. reported women with atypical hyperplasia have nearly a 30% incidence of breast cancer at 25-year follow-up. Women with dense breast tissue have up to a 4-fold increased risk of breast cancer when compared to average-risk women; their cancers are more likely to be mammographically occult. Because multiple cohorts of women are at high risk for developing breast cancer, there has been a movement to develop an abbreviated MRI (abMRI) protocol to expand the availability of MRI screening. Studies on abMRI effectiveness have been promising, with Weinstein et al. demonstrating a cancer detection rate of 27.4/1000 in women with dense breasts after a negative digital breast tomosynthesis. Breast MRI is also used to evaluate the extent of disease as part of preoperative assessment in women with newly diagnosed breast cancer, and to assess a patient’s response to neoadjuvant chemotherapy. This paper aims to explore the current uses of MRI and propose future indications and directions.

// Trevor J. Grant 1 , Anita K. Mehta 1 , Anamika Gupta 1 , Ahmad A.D. Sharif 2 , Kshitij S. Arora 3 , Vikram Deshpande 3 , David T. Ting 3 , Nabeel Bardeesy 3 , Neil J. Ganem 1 , Alexander Hergovich 2 and Anurag Singh 1 1 Department of Pharmacology and Experimental Therapeutics, Center for Cancer Research, Boston University Graduate School of Medicine, Boston, MA, USA 2 University College London, Cancer Institute, London, United Kingdom 3 Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, USA Correspondence to: Anurag Singh, email: asingh3@bu.edu Keywords: KRAS, oncogene dependency, Hippo pathway, pancreatic cancer, kinase Received: July 12, 2017 Accepted: August 27, 2017 Published: September 11, 2017 ABSTRACT Pancreatic ductal adenocarcinomas (PDACs) are highly aggressive malignancies, associated with poor clinical prognosis and limited therapeutic options. Oncogenic KRAS mutations are found in over 90% of PDACs, playing a central role in tumor progression. Global gene expression profiling of PDAC reveals 3-4 major molecular subtypes with distinct phenotypic traits and pharmacological vulnerabilities, including variations in oncogenic KRAS pathway dependencies. PDAC cell lines of the aberrantly differentiated endocrine exocrine (ADEX) subtype are robustly KRAS-dependent for survival. The KRAS gene is located on chromosome 12p11-12p12, a region amplified in 5-10% of primary PDACs. Within this amplicon, we identified co-amplification of KRAS with the STK38L gene in a subset of primary human PDACs and PDAC cell lines. Therefore, we determined whether PDAC cell lines are dependent on STK38L expression for proliferation and viability. STK38L encodes a serine/threonine kinase, which shares homology with Hippo pathway kinases LATS1/2. We show that STK38L expression is elevated in a subset of primary PDACs and PDAC cell lines displaying ADEX subtype characteristics, including overexpression of mutant KRAS. RNAi-mediated depletion of STK38L in a subset of ADEX subtype cell lines inhibits cellular proliferation and induces apoptosis. Concomitant with these effects, STK38L depletion causes increased expression of the LATS2 kinase and the cell cycle regulator p21. LATS2 depletion partially rescues the cytostatic and cytotoxic effects of STK38L depletion. Lastly, high STK38L mRNA expression is associated with decreased overall patient survival in PDACs. Collectively, our findings implicate STK38L as a candidate targetable vulnerability in a subset of molecularly-defined PDACs.

MEK inhibitors have limited efficacy in treating RAS–RAF–MEK pathway–dependent cancers due to feedback pathway compensation and dose-limiting toxicities. Combining MEK inhibitors with other targeted agents may enhance efficacy. Here, codependencies of MEK, TAK1, and KRAS in colon cancer were investigated. Combined inhibition of MEK and TAK1 potentiates apoptosis in KRAS-dependent cells. Pharmacologic studies and cell-cycle analyses on a large panel of colon cancer cell lines demonstrate that MEK/TAK1 inhibition induces cell death, as assessed by sub-G1 accumulation, in a distinct subset of cell lines. Furthermore, TAK1 inhibition causes G2–M cell-cycle blockade and polyploidy in many of the cell lines. MEK plus TAK1 inhibition causes reduced G2–M/polyploid cell numbers and additive cytotoxic effects in KRAS/TAK1-dependent cell lines as well as a subset of BRAF-mutant cells. Mechanistically, sensitivity to MEK/TAK1 inhibition can be conferred by KRAS and BMP receptor activation, which promote expression of NF-κB-dependent proinflammatory cytokines, driving tumor cell survival and proliferation. MEK/TAK1 inhibition causes reduced mTOR, Wnt, and NF-κB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NF-κB transcriptional signature was derived that stratifies primary tumors into three major subtypes: Wnt-high/NF-κB-low, Wnt-low/NF-κB-high and Wnt-high/NF-κB-high, designated W, N, and WN, respectively. These subtypes have distinct characteristics, including enrichment for BRAF mutations with serrated carcinoma histology in the N subtype. Both N and WN subtypes bear molecular hallmarks of MEK and TAK1 dependency seen in cell lines. Therefore, N and WN subtype signatures could be utilized to identify tumors that are most sensitive to anti-MEK/TAK1 therapeutics. Implications: This study describes a potential therapeutic strategy for a subset of colon cancers that are dependent on oncogenic KRAS signaling pathways, which are currently difficult to block with selective agents. Mol Cancer Res; 14(12); 1204–16. ©2016 AACR.

Patients with BRCA-associated triple-negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies the greatest infiltration of immune cells into the tumor was macrophages. Given that objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high-dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wild-type TNBC. Using a preclinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing costimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an antitumor phenotype. We also identified a novel negative feedback mechanism that limits the functionality of the anti-tumor TAMs and is consistent with induction of an immune-suppressive macrophage population. Utilizing transcriptomic, proteomic, and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biologic processes associated with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the preclinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by protumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Anita K. Mehta, Emily M. Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Christina A. Hartl, Nathan T. Johnson, William M. Oldham, Marian Kalocsay, Sarah A. Boswell, Olmo Sonzogni, Constantia Pantelidou, Brett P. Gross, Shawn Johnson, Deborah A. Dillon, Sandro Santagata, Judy E. Garber, Nadine Tung, Elizabeth A. Mittendorf, Gerburg M. Wulf, Geoffrey I. Shapiro, Peter K. Sorger, Jennifer L. Guerriero. PARP inhibition modulates the infiltration, phenotype, and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the antitumor potential of TAMs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A105.

The heterogeneity of the tumor microenvironment may contribute to the lack of durable responses of immunotherapy in breast cancer. To understand factors that contribute to tumor immune cell heterogeneity, we report a detailed analysis and comparison of the immune tumor microenvironment of the autochthonous MMTV-PyMT murine breast cancer model resembling luminal B breast cancer and corresponding syngeneic models. We obtained tumors from MMTV-PyMT mice and used them to generate syngeneic models using either 1E6, 1E5, or 1E4 cells injected into the mammary fat pad of FVB/NJ wild-type mice. When tumors reached 100 mm3, tumors were harvested and quantitative flow-cytometry and NanoString analysis was performed. We have identified that the number of cells inoculated to generate syngeneic tumors significantly influences tumor latency, the tumor immune microenvironment, and the response to immune checkpoint blockade (ICB). Compared to the autochthonous model, the 1E6 and 1E5 model had significantly more tumor-infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and the highest proportion of PD-L1-positive myeloid cells. We found that increased TILs and expression of PD-L1 on myeloid cells were the best predictors of response to PD-L1 or CTLA-4 therapy but that tumor cell expression of PD-L1 and T-cell expression of PD-1 did not correspond to beneficial outcome of treatment. Both the 1E6 and 1E5 models responded to PD-L1 and/or CTLA-4 ICB therapy, whereas the 1E4 and autochthonous models were resistant. These matched sensitive and resistant tumor models represent a unique opportunity to further interrogate the TME in breast cancer. Citation Format: Anita K. Mehta, Jessica A. Castrillion, Alaba Sotayo, Elizabeth A. Mittendorf, Anthony G. Letai, Jennifer L. Guerriero, Emily Cheney. Identifying cellular immune components that correlate with response to immunotherapy in breast cancer using murine models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A81.

Patients with BRCA-associated triple negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T-cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies, the greatest infiltration of immune cells into the tumor was macrophages. Given objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was presumably due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wildtype TNBC. Using a pre-clinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing co-stimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an anti-tumor phenotype. We also identified a novel negative feedback mechanism which limits the functionality of the anti-tumor TAMs, and is consistent with induction of an immune suppressive macrophage population. Utilizing transcriptomic, proteomic and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biological processes associate with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the pre-clinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by pro-tumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Jennifer L Guerriero, Anita K Mehta, Emily M Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Olmo Sonzogni, Constantia Pantelidou, Christina A Hartl, William M Oldham, Nathan T Johnson, Sarah A Boswell, Marian Kalocsay, Matthew J Berberich, Sholin Mei, Dan Wang, Shawn Johnson, Brett Gross, Deborah A Dillon, Mikel Lipschitz, Evisa Gjini, Scott Rodig, Sandro Santagata, Judy E Garber, Nadine Tung, Peter Sorger, Geoffrey I Shapiro, Gerburg M Wulf, Elizabeth A Mittendorf. PARP inhibition modulates the infiltration, phenotype and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the anti-tumor potential of TAMs [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-01.

Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8(+) T cell infiltration and activation in vivo, and that CD8(+) T cell depletion severely compromises anti-tumor efficacy. Olaparib-induced T cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-deficient compared to HR-proficient TNBC cells and in vivo models. CRISPR-knockout of STING in cancer cells prevents proinflammatory signaling and is sufficient to abolish olaparib-induced T cell infiltration in vivo. These findings elucidate an additional mechanism of action of PARP inhibitors and provide rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC.

KRAS mutant non-small cell lung cancer (NSCLC) may be classified into epithelial or mesenchymal subtypes. Mesenchymal NSCLCs and associated “KM” cell lines are generally less responsive than their epithelial counterparts to inhibition of the RAS pathway; identifying alternative networks that promote mesenchymal NSCLC survival may lead to more effective treatments for this subtype. Non-coding microRNA (miRNA) signatures can stratify tumors into diverse molecular subtypes. By regulating numerous targets in cancer-associated signaling pathways, miRNAs can function as tumor suppressors or oncogenes. In particular, some miRNAs regulate the epithelial-mesenchymal transition (EMT). Here, we derived an EMT-related miRNA signature by profiling the abundance of miRNAs in a panel of epithelial (KE) or mesenchymal (KM) KRAS mutant NSCLC cell lines. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family, which can promote tumor suppression by inhibiting EMT. Reconstituting KM cells with one of these miRNAs, miR-124, disrupted autophagy and decreased cell survival by suppressing the abundance of p62, an adaptor for selective autophagy and regulator of the transcription factor NF-κB. Suppression of p62 by miR-124 correlated with reduced abundance of the autophagy activator Beclin 1, the ubiquitin ligase TRAF6 and the NF-κB subunit RELA/p65. Abundance of miR-124 inversely correlated with expression of BECN1 and TRAF6 in patient NSCLC samples. These findings identify a role for miR-124 in regulating cell survival networks in a specific subtype of KRAS mutant NSCLC cell lines, which might lead to improved subtype-selective therapeutic strategies for patients.

KRAS mutant non–small cell lung cancer (NSCLC) may be classified into epithelial or mesenchymal subtypes. Despite having the same “driver” mutation, mesenchymal NSCLCs are less responsive than are epithelial NSCLCs to inhibition of the RAS pathway. Identifying alternative networks that promote survival specifically in mesenchymal NSCLC may lead to more effective treatments for this subtype. Through their numerous targets in cellular signaling pathways, noncoding microRNAs (miRNAs) often function as tumor suppressors or oncogenes. In particular, some miRNAs regulate the epithelial-mesenchymal transition (EMT). We derived an EMT-related miRNA signature by profiling the abundance of miRNAs in a panel of epithelial (KE) or mesenchymal (KM) KRAS mutant NSCLC cell lines. This signature revealed a number of suppressed miRNAs in KM cell lines, including members of the miR-200 family, which can suppress tumor progression by inhibiting EMT. Reconstituting KM cells with one of these miRNAs, miR-124, disrupted autophagy and decreased cell survival by reducing the abundance of p62, which is both an adaptor for selective autophagy and a regulator of the transcription factor nuclear factor κB (NF-κB). Suppression of p62 by miR-124 correlated with reduced abundance of the autophagy activator beclin 1 (BECN1), the ubiquitin ligase TRAF6, and the NF-κB subunit RELA/p65. The abundance of miR-124 inversely correlated with the expression of BECN1 and TRAF6 in patient NSCLC samples. These findings reveal how the loss of miR-124 promotes cell survival networks in the aggressive mesenchymal subtype of KRAS mutant NSCLC, which might lead to improved subtype-selective therapeutic strategies for patients.

The purpose of this section is to provide a detailed review of high-risk lesions and their associated risk of upgrade at surgical excision in order to guide in management and appropriate risk reduction strategies. “High-risk” breast lesions refer to an eclectic group of histologic abnormalities associated with an increased risk of breast cancer. Current data show a vast disparity in upgrade rates upon surgical excision for high-risk lesions, which leads to confusing and often conflicting recommendations. There has been suggestion in the media that breast biopsies lead to “over-diagnosis” and unnecessary breast surgeries. However, the goal of breast imagers is to detect early cancers, and in doing so, the recommendation for surgical excision of the appropriate high-risk lesions is necessary. In managing high-risk lesions we must balance the opportunity to diagnose early, curable breast cancer by recommending surgical excision with prudent and conservative management along with careful radiologic and pathologic correlation.

Oncogenic NRAS mutations are found in 20 percent of sporadic melanoma cases. Mutant NRAS activates the RAF/MEK/ERK mitogen-activated protein kinase (MAPK), PI3K/AKT and RAC1/PAK kinase signaling pathways, which cooperate to drive melanomagenesis. The complexity of the NRAS signaling network presents a challenge to develop effective therapeutics for NRAS mutant melanoma. Many inhibitors of NRAS-regulated signaling pathways, such as BRAF and MEK/ERK inhibitors are ineffective in this setting. Furthermore, direct NRAS inhibition is pharmacologically challenging. It is likely that combined inhibition of two or more nodes in the NRAS signaling network will be effective in treating NRASmutant melanoma. We have used whole-genome transcriptional profiling coupled with phenotypic characterization of NRAS dependency to derive an NRAS dependency gene expression signature. The signature reveals two key features: (1) NRAS dependency is associated with an kinase-regulated inflammatory gene expression program and (2) expression of the melanocyte lineage factor MITF is strongly associated with NRAS-independent cell proliferation. Based on this knowledge, we have identified combinatorial therapeutic modalities to target either NRAS-dependent or NRAS-independent melanoma subtypes. NRAS-dependent melanoma cell lines undergo apoptotic cell death upon combined inhibition of PAK kinases and Bcl2/Bcl-XL proteins. Conversely, NRAS-independent cell lines are sensitive to the apoptotic effects of combined CDK2 and ERK kinase inhibition. Notably, CDK2 is an MITF transcriptional target gene that mediates a feed-forward loop to potentiate MITF activation. Taken together, our studies highlight phenotypic variations in NRAS mutant melanomas that can be exploited to identify subtype-specific therapeutic targets for NRAS mutant melanoma. Citation Format: Anita K. Mehta, Kevin Hua, Jayant Gadrey, Angelina Fuller, Anurag Singh. Molecular and pharmacological characterization of mutant NRAS dependency in melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-090.

Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8+ T cell infiltration and activation in vivo, and that depletion of CD8+ T cells severely compromises anti-tumor efficacy. Olaparib-induced T cell recruitment is mediated through activation of the STING/TBK1/IRF3 pathway in tumor and dendritic cells and is more pronounced in HR-deficient compared to HR-proficient TNBC cells. CRISPR-knockout of STING in cancer cells prevents type I IFN production and is sufficient to abolish PARP inhibitor-induced T cell infiltration in vivo. These findings elucidate a novel mechanism of action of PARP inhibitors and provide mechanistic rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. Citation Format: Constantia Pantelidou, Olmo Sonzogni, Mateus De Oliveira Taveira, Anita K. Mehta, Dan Wang, Aditi Kothari, Michelle K. Li, Tanvi H. Visal, Jennifer L. Guerriero, Gerburg M. Wulf, Geoffrey I. Shapiro. PARP inhibitor efficacy depends on CD8+ T-cell recruitment via the STING pathway in BRCA-deficient models of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4490.

Background KRAS mutant non-small cell lung cancers (NSCLC) are molecularly and histologically diverse. Epithelial-like cells are more KRAS dependent, whereas mesenchymal-like cells are less KRAS dependent. These two subtypes are designated KE (epithelial) and KM (mesenchymal), respectively. A KE versus KM subtype transcriptional signature reveals specific modes of KRAS dependent survival signaling in the KE subtype. This KRAS dependency signature is significantly enriched with predicted microRNA (miRNA) target genes of miR-205 and miR-34b/c. MiRNAs can function as tumor suppressors by coordinately regulating multiple oncogenic signaling pathways. The role of deregulated miRNA function in mediating the survival of KM versus KE NSCLC cells has not been investigated to date. Methods Differential miRNA expression in KRAS mutant cell lines was determined using Taqman low-density qPCR arrays (TLDA). Functional miRNA reconstitution experiments of downregulated miRNAs were performed in a panel of KM cell lines. Effects on apoptosis and autophagy were performed by Western blotting, immunofluorescence and live cell microscopy and caspase assays. The molecular targets of miR-124 were computationally identified by TargetScan or miRWalk and experimentally verified using 3’UTR luciferase-based assays. Functional rescue of miRNA-dependent cell viability defects was determined by ectopic predicted target gene expression. Results Comparison of KE to KM cells yielded a KE-KM miRNA subtype classifier/signature. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family. MiR-200 and miR-205 reconstitution in KM cells modulated epithelial plasticity by Zeb1 protein suppression and increased E-cadherin levels. Reconstitution of miR-124, miR-625 and miR-518-3p in KM cells caused pronounced loss of cell viability. Furthermore, miR-124 caused autolysosome maturation defects. We identified SQSTM1/p62, TRAF6 and RELA/p65 as key predicted targets of miR-124. MiR-124 reconstitution in KM cells caused decreased p62, TRAF6 and p65 protein levels. The effect of miR-124 on p62 expression was verified using a Luciferase-p62-3’UTR reporter construct. Overexpression of p62 in KM cells rescued the cell viability defects caused by miR-124. Conclusion These studies implicate miR-124 as a context-dependent tumor suppressor miRNA in KM subtype cells. MiR-124 directly suppresses expression of SQSTM1/p62 to promote defects in autolysosome maturation. In parallel, miR-124 suppresses RELA/ p65 and in some cases, TRAF6, to alter expression levels of several cytokines. Thus, miR-124 coordinately regulates autophagy and inflammation to disrupt the finely-tuned balance between pro and anti-inflammatory signals, resulting in cytotoxic effects in a specific subtype of mesenchymal-like KRAS mutant NSCLC cells. Citation Format: Anita K. Mehta, Kevin Hua, William Whipple, Mihn-Thuy Nguyen, Rushika M. Perera, Johanns Haybaeck, Joanne Weidhass, Jeffrey Settleman, Anurag Singh. MiR-124 suppresses p62 and p65/NFkB to regulate autophagy, inflammation and cell death in KRAS mutant mesenchymal NSCLC cells [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 2524. doi:10.1158/1538-7445.AM2017-2524

Eukaryotic gene expression is a complicated process primarily regulated at the levels of gene transcription and mRNA translation. The latter involves four main steps: initiation, elongation, termination and recycling. Translation regulation is primarily achieved during initiation which is orchestrated by 12 currently known eukaryotic initiation factors (eIFs). Here, we review the current state of eIF research and present a concise summary of the various eIF subunits. As eIFs turned out to be critically implicated in different oncogenic processes the various eIF members and their contribution to onset and progression of cancer are featured.

Background Non-small cell lung cancer (NSCLC) is derived from broncho-alveolar epithelial cells. NSCLCs frequently harbor mutations in the KRAS oncogene, yet only a subset of NSCLC cell lines depend on KRAS for sustained survival signaling. Cells that are more epithelial are highly KRAS dependent, whereas mesenchymal-like cells are KRAS independent for cell survival signaling. KRAS mutations are commonly associated with resistance to anti-cancer therapeutics. An important drug resistance mechanism is altered cellular plasticity, via a process known as epithelial-to-mesenchymal transition (EMT). Here, we aimed to identify and elucidate a KRAS dependent microRNA (miRNA) signaling network in epithelial cancer cells that modulates malignant growth and proliferation as well as cellular plasticity in NSCLC. Methods Differential miRNA expression in 6 KRAS mutant NSCLC cell lines (3 KRAS independent versus 3 KRAS dependent) was determined using Taqman low-density qPCR arrays (TLDA). To explore the functional relevance of differentially regulated miRNAs, gain-of-function studies by reconstitution experiments were performed using miRNA mimics in a panel of six KRAS independent cell lines. The molecular targets of mir-124-2 were computationally identified by TargetScan or miRWalk and experimentally verified using 3'UTR luciferase-based assays. Functional rescue of cell viability defects was determined by forced expression of GFP-p62/Sqstm1 in KRAS independent lung cancer cell lines. Results We identified a distinct differential miRNA expression profile in KRAS dependent versus KRAS independent NSCLC cells. This KRAS dependency miRNA signature included members of the mir-200 family, mir-34c and mir-205. Mir-200 and mir-205 reconstitution in KRAS independent cells modulated epithelial plasticity by down regulating Zeb1 protein expression and, thereby, increasing E-cadherin levels. Ectopic introduction of a subset of miRNAs in KRAS independent cells caused a consistent and pronounced loss of cell viability in all cell lines tested, including mir124, mir-625 and mir-518-3p. Ectopic expression of mir124 in KRAS independent cells caused cell viability defects by inducing apoptosis and autophagy. Using bioinformatics databases, we isolated p62/Sqstm1 and REL-A as key predicted targets of mir124-2. Mir-124-2 reconstitution in KRAS independent NSCLC cell lines caused decreased p62/Sqstm1 and REL-A protein expression levels. The effect of mir-124 on p62/Sqstm1 expression was verified using a p62-3'UTR-Luciferase construct. Overexpression of GFP-p62 in KRAS independent cells rescued the cell viability defects by mir124-2. Conclusion MiR-124 expression is down regulated in KRAS independent NSCLC cells. Reintroduction of mir124-2 induces cell viability defects in part, by targeting p62/Sqstm1 and REL-A. We conclude that, mir-124 down regulates p62/Sqstm1 expression to activate autophagic flux. This leads to down regulation of the NFκB pathway and subsequent reduction in expression of pro-survival genes such as IL-6 and IL-1β. Taken together, mir124-2 acts as a tumor suppressor in KRAS mutant NSCLC cells and provides a mechanistic link between activation of autophagy and control of proinflammatory signaling in NSCLC cell survival. Overexpression of mir124-2 might be of potential therapeutic value for the treatment of KRAS mutant NSCLC. Citation Format: ANITA K. MEHTA, Kevin Hua, William Whipple, Minh-Thuy Nguyen, Stephanie Bartley, Margaret McGuiness, Jeff Settleman, Joanne Weidhaas, Anurag Singh. A KRAS dependency microRNA signature reveals a p62/Sqstm1-centered autophagy network regulated by mir-124. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C02.

The KRAS protooncogene is mutated in 40 to 50% of colon cancers. In an effort to identify strategies to treat KRAS mutant colon cancers, we previously implicated the TGF-β activated kinase (TAK1) as a candidate therapeutic target that promotes the survival of KRAS dependent cancers. In follow-up studies, we have explored and investigated the detailed mechanistic basis for TAK1 mediated survival signaling in KRAS dependent cancer cells. Using proteomic and transcriptomic analyses of proinflammatory signaling mediators, we have uncovered complex autocrine/paracrine signaling loops that are constitutively activated in KRAS dependent colon cancer cells. A central mediator of these signaling loops is the BMP7-BMP receptor (BMPR1A) pathway, which functions coordinately with oncogenic KRAS to drive TAK1 and NF-κB mediated transcriptional upregulation of proinflammatory cytokines such as GM-CSF, CCL5/RANTES and IL-8. Conversely, a number of cytokines and cytokine regulators are negatively regulated by KRAS-BMPR signaling interactions, including CXCL9/MIG and IL1RN. We previously showed that TAK1 inhibition with a small molecule agent, 5Z-7-oxozeaenol promotes apoptosis in colon cancer cells. We have now determined that 5Z-7-oxozeaenol, in addition to irreversibly inhibiting TAK1 kinase activity, also transiently inhibits the MEK kinase. Therefore, combined TAK1/MEK inhibition explains the potent killing effects that we have observed in KRAS dependent colon cancer cells. To test this empirically, we have used 2 selective kinase inhibitors targeting each respective kinase, AZ-TAK1 and AZD6244 to show either single agent can induce apoptotic cell selectively in KRAS dependent cells, with TAK1 inhibition resulting in stronger killing effects. Importantly, treatment of KRAS dependent colon cancer cells with combinations of AZ-TAK1 and AZD6244 results in additive killing effects, revealing a potential therapeutic strategy for KRAS dependent cancers in the clinic. Mechanistically, MEK and TAK1 converge on the control of NF-κB and canonical Wnt-dependent transcriptional activities. Surprisingly, we find that NF-κB and Wnt signaling mutually antagonize each other in terms of cytokine expression to create a finely-tuned balance between pro-death and pro-survival signals. We hypothesize that, as a consequence, these balanced signals allow for both efficient maintenance of tumor cell survival and as well as for communication with stromal components in the tumor microenvironment. Thus, KRAS, MEK or TAK1 blockade results in a tipping of the balance to favor pro-death signals. Citation Format: Kelsey L. McNew, William J. Whipple, Anita K. Mehta, Trevor Grant, Anurag Singh. MEK and TAK1 signaling interactions coordinately regulate inflammation and apoptosis in KRAS dependent colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3188. doi:10.1158/1538-7445.AM2015-3188

Background: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, hepatocyte ballooning and cytoplasmic protein aggregates termed Mallory-Denk bodies (MDBs). MDBs are generally composed of misfolded keratin (K) 8, 18 and in part p62 and ubiquitin. It is well established that K aggregates are critical for MDB formation, thus Ks are associated with SH which is one of the main preconditions for the development of liver cirrhosis and hepatocellular carcinoma (HCC). Aims: In this study we aimed to clarify the cellular and molecular mechanisms of relative K8 excess over K18 on hepatocarcinogenesis in mice and its functional and clinical implication in human alcoholic and non-alcoholic SH (ASH/NASH)-induced liver cancer. Methods: 18 month-old krt18-deficient (krt18-/-129P2/OlaHsd background) mice were investigated for the incidence of SH and SH-induced liver tumors by radiology, macroscopy, histology, immunohistochemistry, gene expression, lipidome analysis, and immunoblotting. Results: Aged krt18-/- mice developed the entire morphological spectrum of SH whereas aged wild-type (wt) mice showed simple steatosis. Aminotransaminase levels were also elevated in aged krt18-/- mice. Interestingly, 64% of male and 25% of female 18 month-old krt18-/- mice developed liver tumors revealing morphological and genetic features of HCC whereas 45% of male and 16% of female krt18+/- mice and 42% of wt mice developed HCC. Conclusion: Aged krt18-/- mice represent a novel spontaneous SH and SH associated HCC model with significant gender differences revealing features related to human HCC. Therefore, variations of hepatocellular K18 seem to have an effect on the susceptibility regarding SH and SH-induced HCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A38. Citation Format: Anita Kuldeep Mehta, Kira Bettermann, Eva M. Lederer, Christina Ernst, Sonja M. Kessler, Kensuke Kojima, Xintong Chen, Yujin Hoshida, Bryan C. Fuchs, Vendula Svendova, Michael G. Schimek, Monika Mach, Michael R. Speicher, Tatjana Stojakovic, Thomas M. Magin, Pavel Strnad, Michael Trauner, Alexandra K. Kiemer, Snorri S. Thorgeirsson, Michael Karin, Josep M. Llovet, Kurt Zatloukal, Helmut Denk, Carolin Lackner, Johannes Haybaeck. Steatohepatitis-associated hepatocellular carcinoma: Evidence of a keratin-based disease. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A38.