bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B52: HIF-ZMYND8-BRD4 axis mediates breast cancer progression and metastasis
Authors: Chen Yan , Zhang Bo , Bao Lei , Jin Lai , Yang Mingming , Peng Yan , Wang Jennifer , Wang Chenliang , Zou Xuan , Wang Yingfei , Luo Weibo
Published: August 2018

Metastatic breast cancer is lethal and incurable. The major obstacle to ending this aggressive disease is the absence of definitive prognostic biomarker and effective therapeutic target. Epigenetic dysregulation plays a crucial role in breast cancer metastasis. However, how epigenetic dysregulation under tumor hypoxia stimulates breast cancer progression and metastasis remains unknown.

We recently identified the epigenetic reader ZMYND8 as a novel HIF target gene by analyzing expression changes of 720 epigenetic genes in three microarray gene expression and mRNA sequencing datasets. Analysis of a human breast cancer tissue microarray revealed that ZMYND8 is highly expressed in invasive breast tumors and high levels of ZMYND8 are significantly correlated with poor clinical outcomes in patients with breast cancer. ZMYND8 depletion significantly suppresses colony formation, invasion, and migration of breast cancer cells in vitro, and inhibits breast tumor growth and metastasis in mice. To figure out the underlying mechanism, we performed co-immunoprecipitation, chromatin immunoprecipitation, and quantitative PCR assays and found that ZMYND8 interacts with HIF complex and acetyl lysine 16 of histone H3 at the hypoxia response elements (HREs) to stimulate the expression of HIF target genes LOX, AGR2, AQP1, and VEGFA. HIF is required for ZMYND8-mediated breast cancer growth and metastasis in mice. We further found that p300 binds and acetylates ZMYND8 in breast cancer cells. Acetylated ZMYND8 interacts with the bromodomains of BRD4 and recruits BRD4 to the HREs to stimulate RNA polymerase II phosphorylation and subsequent transcriptional elongation of HIF target genes. ZMYND8 acetylation is necessary and sufficient for breast tumor growth and metastasis in vitro and in mice. Together, these findings indicate that p300-acetylated ZMYND8 mediates breast cancer progression and metastasis by BRD4-dependent activation of HIF.

In summary, ZMYND8 represents a positive-feedback mechanism that amplifies HIF-mediated breast cancer progression and metastasis, and may be a potential epigenetic target for the prognosis and treatment of breast cancer.

Citation Format: Yan Chen, Bo Zhang, Lei Bao, Lai Jin, Mingming Yang, Yan Peng, Jennifer Wang, Chenliang Wang, Xuan Zou, Yingfei Wang, Weibo Luo. HIF-ZMYND8-BRD4 axis mediates breast cancer progression and metastasis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B52.
Abstract B59: Multiparametric imaging to evaluate human and mouse breast carcinoma in situ
Authors: Sinha Vidya C. , Yang Fei , Rinkenbaugh Amanda L. , Zhou Xinhui , Zhang Xiaomei , Wistuba Ignacio I. , Piwnica-Worms Helen
Published: August 2018

Our overall goal is to distinguish aggressive from indolent breast cancers by characterizing determinants of breast cancer progression. The detection of early-stage (in situ) neoplastic lesions in the breast has risen dramatically due to widespread mammography. Early detection represents a significant opportunity to intervene and halt the progression of these early lesions to invasive breast cancer. However, our understanding of the natural history of in situ lesions remains limited. Here, we describe our efforts to characterize the microenvironment of DCIS in patient samples and mouse models using a combination of traditional and novel multiplexed immunostaining methods. In particular, we present our early work on applying multispectral fluorescence imaging as well as highly parametric imaging mass cytometry to describe the immune cells surrounding and infiltrating DCIS, with focus on the interaction of these immune cells with each other and the incipient invasive tumor cells. Future studies will be directed at determining if the immune microenvironment of DCIS can identify patients at risk for progression or recurrence so that these patients can be appropriately managed upfront.

Citation Format: Vidya C. Sinha, Fei Yang, Amanda L. Rinkenbaugh, Xinhui Zhou, Xiaomei Zhang, Ignacio I. Wistuba, Helen Piwnica-Worms. Multiparametric imaging to evaluate human and mouse breast carcinoma in situ [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B59.
Abstract B60: Omics profiling of CDK4/6 inhibitors reveals functionally important secondary targets of abemaciclib
Authors: Mills Caitlin E. , Hafner Marc , Subramanian Kartik , Chen Chen , Boswell Sarah A. , Everley Robert A. , Juric Dejan , Sorger Peter K.
Published: August 2018

The recent introduction of small-molecule inhibitors of cyclin-dependent kinases (CDK) 4/6 to the clinic has improved the treatment of hormone receptor-positive breast cancer, and shown promise in other malignancies. The three clinically used CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, are reported to be broadly similar, although recent data suggest that abemaciclib has distinct single-agent activity in patients and a unique toxicity profile. In vitro profiling of the CDK4/6 inhibitors has shown abemaciclib to be more potent on target, but less selective than either palbociclib or ribociclib. We sought to systematically investigate the functional consequences of abemaciclib’s polypharmacology. We used molecular and phenotypic profiling by mRNA sequencing, mass spectrometry-based proteomics, and GR-based dose-response assays to characterize the mechanisms of action of the three CDK4/6 inhibitors. Inhibition of CDKs other than CDK4/6 by abemaciclib resulted in an arrest in the G2 phase of the cell cycle, a prolonged inhibition of growth, and cytotoxicity, even in retinoblastoma protein (pRb)-deficient cells. Abemaciclib elicited unique molecular responses at clinically achievable concentrations that are likely to be therapeutically advantageous and prevented cross-resistance with ribociclib and palbociclib. More generally, we propose that multi-omic approaches are required to fully elucidate the spectrum of targets relevant to drug mechanisms of action in cells, and we expect such understanding to assist in stratifying patient populations and in ordering sequential therapies when resistance arises.

Citation Format: Caitlin E. Mills, Marc Hafner, Kartik Subramanian, Chen Chen, Sarah A. Boswell, Robert A. Everley, Dejan Juric, Peter K. Sorger. Omics profiling of CDK4/6 inhibitors reveals functionally important secondary targets of abemaciclib [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B60.
Abstract B69: The effects of patient-physician relationships on perceptions of breast cancer treatment in African American women
Authors: Lauray Alexandria N. , Bigatti Silvia
Published: August 2018

According to Indiana.gov, 4,400 new cases of breast cancer are diagnosed in Indiana annually. “During 2008 to 2012, the mortality rates of African American breast cancer patients were almost 40% higher than Caucasian Americans” (Cancer.org), which sheds light on the health disparities African Americans face in the United States. Disparities among cancer patients have not only led to African American women (AAW) being diagnosed in later stages of this disease than their counterparts and thus raising their mortality rates, but AAW tend to be diagnosed with more aggressive forms of cancer such as triple-negative breast cancer, which occurs in 10% to 20% of patients. AAW face many barriers today: limited access to screening services, lack of quality screening equipment, limited access to treatment services, and an unspoken distrust of health care providers. Research indicates that during their breast cancer treatment trajectory, AAW experience delay in initiating chemotherapy after surgery, less satisfaction with treatment, increased symptoms, and lower participation in clinical trials (Mcarthy). Despite this, there is no study that has followed and assessed AAW during the treatment sessions to examine challenges in their lives and their impact. Studies of AAW who have survived breast cancer suggest the need to look not only at factors within the medical care system, but well beyond it into the everyday lives of these women and the resources available to them through social networks and other means to overcome challenges. This project explores themes in the baseline and exit interviews of 38 participants. In their initial interview, we asked questions related to support system, their views of treatment going into the process, and the quality of care that they feel they have received. At the end of treatments, we ask about patient-physician relationships and how the women perceived their care. Women in the study reported the need to readdress concerns with physicians. Patient-physician relationships among AAW diagnosed with breast cancer have been strained and have had a negative impact on patient satisfaction with care.

Note: This abstract was not presented at the conference.

Citation Format: Alexandria N. Lauray, Silvia Bigatti. The effects of patient-physician relationships on perceptions of breast cancer treatment in African American women [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B69.
Abstract IA26: Clinical development of PI3K/AKT pathway inhibitors in breast cancer
Authors: Derynck Mika
Published: August 2018

Targeting the PI3K/AKT/mTOR pathway has been a focus for drug development since the discovery of PI3K-activating mutations in p110α over 10 years ago. Breast cancer has been a primary indication for development of this class of drugs because of the high prevalence of these mutations, up to 45% in hormone receptor-positive breast cancer, and the synergy observed in combinations with endocrine therapies. An mTOR inhibitor, everolimus, was approved in breast cancer in combination with exemestane in 2012, indicating the synergy between ER and PI3K pathway translates to the clinic. Apitolisib, a dual PI3K pan-isoform and mTOR inhibitor, has discontinued development due to toxicities, despite compelling preclinical activity across multiple tumor types regardless of PI3K and PTEN status. Pictilisib, a pan-isoform PI3K inhibitor, has shown some evidence of activity, but in phase II studies has demonstrated that the activity is limited by dose intensity. Buparlisib, a pan-isoform PI3K inhibitor, has demonstrated efficacy in a large phase III study, but was not deemed sufficiently active relative to the toxicity in HR+ breast cancer to be submitted for regulatory approval. Second-generation PI3K and AKT inhibitors with higher selectivity and mutant specificity are in clinical development and hope to thread a better therapeutic index by dosing higher for more profound inhibition, and/or selectively inhibiting mutated forms of PI3K. Taselisib (GDC-0032) is a beta-sparing and PI3K-mutant selective inhibitor that leads to degradation of only the mutant form of PI3K and is currently in phase III. More recently, taselisib met the primary endpoint of improved response rates in a randomized, neoadjuvant phase II trial, LORELEI. Alpelisib is an alpha-selective PI3K inhibitor that is also in phase III. Recent data with ipatasertib, an AKT inhibitor, has demonstrated better tolerability and efficacy in randomized phase II trials of both prostate cancer and triple-negative breast cancer, where the PI3K/AKT signaling pathway is activated by either loss of PTEN or PI3K mutations, and may offer a better therapeutic index given its unique mechanism of action and place in the PI3K signaling pathway. Overall, the PI3K/AKT pathway is still considered an important pathway to target for certain cancers; however, target selectivity, dosing, and the right combination are critical to find a therapeutic index to provide a good option for patients.

Citation Format: Mika Derynck. Clinical development of PI3K/AKT pathway inhibitors in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA26.
Abstract IA31: What limits the response of breast cancer to immunotherapy?
Authors: Loi Sherene
Published: August 2018

Checkpoint blockade has been evaluated successfully in the classically immunogenic solid tumor types such as melanoma and renal cell carcinoma, as well as in hematologic malignancies. While a large degree of T-cell infiltrate has been observed in primary breast cancers, particularly triple-negative and Her2-positive subtypes, efficacy with checkpoint blockade as monotherapy has been modest so far in studies in advanced disease.

Given that higher levels of T-cell infiltrate have been associated with improved prognoses and response to cytotoxic chemotherapy, my lab has hypothesized that these T cells would have functional characteristics that would be important in immunosurveillance of breast cancer. We have characterized these T cells using methods such as multiplex immunohistochemistry, bulk gene expression, and flow cytometry, as well as single-cell transcriptomic sequencing in order to better characterize these T cells. We have also observed lower levels of immune infiltrate in samples from metastatic breast cancer lesions, suggesting that the primary disease setting may be the most amenable to checkpoint inhibitor approaches. Data currently support that line of therapy in the advanced breast cancer setting; tumor burden and T-cell infiltrate predict for higher response rates to PD-1 blockade as monotherapy.

Finally, given that most patients with advanced breast cancer have a “noninflamed” tumor microenvironment, my lab has been evaluating how therapeutics can modulate the immune microenvironment. Certain agents such as MEK inhibitors as well as combination targeted therapies can potentially increase tumor immunogenicity as well as synergize well with immunotherapies. These combinatorial approaches may serve to increase the response rate of immunotherapy in breast cancer patients as well as prolong duration of response in patients treated with new therapeutic agents.

Citation Format: Sherene Loi. What limits the response of breast cancer to immunotherapy? [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA31.
Abstract 48: Acid ceramidase inhibition: A targeted therapy for acute myeloid leukemia
Authors: Pearson Jennifer M. , Tan Su-Fern , Sharma Arati , Fox Todd E. , Abad Jose Luis , Fabrias Gemma , Claxton David F. , Feith David J. , Kester Mark , Loughran Thomas P.
Published: August 2018

Acute myeloid leukemia (AML) is a disease characterized by uncontrolled proliferation of immature myeloid cells in the blood and bone marrow. Patient 5-year survival rate is only 26%, and there have been no significant treatment advances for decades. Therefore, there is an urgent need to identify novel therapeutic targets. We have previously shown that acid ceramidase (AC) is upregulated in AML and high AC activity correlates with poor patient survival. We continue to validate AC as a potential therapeutic target in AML through inhibitor screening and characterization. We utilize viability assays, enzyme activity assays, Western blotting, qPCR, lipidomics, and flow cytometry to examine the effects of inhibitors on AML cell lines, patient samples, and normal controls. We have found that a newly developed selective AC inhibitor, SACLAC, significantly reduces viability of AML cells with an EC50 of approximately 3 μM, with limited effect on normal cells at an EC50 of approximately 13 μM. Treatment of AML cell lines with SACLAC effectively blocks AC activity, induces a drastic decrease of sphingosine 1-phosphate, and a 2.5-fold increase in total ceramide levels. Ongoing studies continue to explore the mechanistic basis for the loss of viability. These data support the investigation of AC as a therapeutic target in AML and define SACLAC as a potent and selective inhibitor that presents promise for preclinical studies and future clinical development.

Citation Format: Jennifer M. Pearson, Su-Fern Tan, Arati Sharma, Todd E. Fox, Jose Luis Abad, Gemma Fabrias, David F. Claxton, David J. Feith, Mark Kester, Thomas P. Loughran, Jr.. Acid ceramidase inhibition: A targeted therapy for acute myeloid leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 48.
Abstract IA19: Directing blood cancer therapy with mitochondrial BH3 profiling
Authors: Letai Anthony G.
Published: August 2018

Connecting the right drugs to the right tumor is the fundamental job of cancer precision medicine. While there is a tendency to equate precision medicine with genomics, we are surrounded by examples where very effective modern targets are deployed effectively in disease without genomic guidance. One example is in chronic lymphocytic leukemia (CLL) where there have been several recent FDA approvals for drugs targeting CD20, PI3K, BTK, and BCL-2. Combinations of these highly active agents will likely dramatically alter practice, yet their discovery and assignment to CLL was not guided by genomics, as CLL generally lacks genetic alterations in any of these pathways. Instead, use in CLL was guided by the identification of vulnerabilities that had more to do with lineage than with genetics. In this talk, I will describe how my laboratory is using a probe of apoptotic signaling at the mitochondrion to identify drugs that provoke apoptotic signaling in cancer cells. This approach, called “BH3 profiling,” has so far been effective in guiding clinical trials of the BCL-2 inhibitor venetoclax in CLL and acute myelogenous leukemia. I will describe how we can deploy this tool as a predictive biomarker or as a discovery tool to directly identify drug vulnerabilities of cancer cells.

Citation Format: Anthony G. Letai. Directing blood cancer therapy with mitochondrial BH3 profiling [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA19.
Abstract A09: Rational combinational therapy with PARP and BRD4 inhibitor in ovarian cancer
Authors: Sun Chaoyang , Yin Jun , Fang Yong , Mills Gordon B.
Published: August 2018

Poly(ADP-ribose) polymerase inhibitors (PARPi) are selectively active in cells with homologous recombination (HR) deficiency (HRD) caused by mutations in BRCA1/2 and other pathway members. We thus sought small-molecule inhibitors that induce HRD in HR competent cells to identify approaches to induce synthetic lethality with PARPi and extend the utility of PARPi. We demonstrate that inhibition of bromodomain containing 4 (BRD4) induces HRD and sensitizes cells across multiple tumor lineages to PARPi regardless of BRCA1/2, TP53, RAS, or BRAF status.

Note: This abstract was not presented at the conference.

Citation Format: Chaoyang Sun, Jun Yin, Yong Fang, Gordon B. Mills. Rational combinational therapy with PARP and BRD4 inhibitor in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A09.
Abstract A67: Effects of the atypical PKC-ι inhibitor ICA-1 on ovarian cancer proliferation and survival
Authors: Smalley Tracess , Acevedo-Duncan Mildred
Published: August 2018

Ovarian cancer is the most lethal gynecologic cancer diagnosis. There is no efficient screening process for ovarian cancers and the average stage of diagnosis is stage III. Atypical PKCs (PKC-ζ and PKC-ι/λ) have been shown to be overexpressed in various malignant cells lines and to be linked to pathways for cellular proliferation and survival. Estrogen is also highly linked to carcinogenesis. In this investigation, ovarian cell lines (HEY, COV644, T80) were treated with the atypical PKC-ι inhibitor ICA-1 in two different systems, with and without estrogen, and assayed to determine the effects on proliferation and cellular survival. These assays included protein quantification, cell proliferation, and wound healing. Our preliminary data suggest that PKC-ι is a novel target in carcinogenesis and inhibition of this protein decreases the rate of proliferation. Our results also suggest that estrogen has a great effect on cell proliferation, even in estrogen receptor-negative cells.

Citation Format: Tracess Smalley, Mildred Acevedo-Duncan. Effects of the atypical PKC-ι inhibitor ICA-1 on ovarian cancer proliferation and survival. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A67.
Abstract B15: A cancer-specific detection of serum CA125 improves differential diagnosis of epithelial ovarian cancer from benign conditions
Authors: Huhtinen Kaisa , Gidwani Kamlesh , Kekki Henna , Hynninen Johanna , Salminen Liina , Perheentupa Antti , Poutanen Matti , Auranen Annika , Grenman Seija , Lamminmäki Urpo , Carpén Olli , Pettersson Kim
Published: August 2018

Introduction: We have developed an assay that distinguishes CA125 originating from malignant and benign sources. The assay combines conventional immunoassay with the detection of carbohydrates differentially glycosylated in epithelial ovarian cancer (EOC). These glycans can be detected using carbohydrate-reactive lectins. Our recent data showed that CA125 detection using MGL-lectin improves the discrimination of cancerous CA125 from CA125 of endometriosis patients (Gidwani et al., Clin.Chem 2016). The current study extends CA125MGL measurements to a broader sample set and longitudinal collections during EOC treatment and follow-up.

Methods: MGL lectin coated onto fluorescent europium-chelate-doped 97-nm nanoparticles was used for detection of the CA125 that was captured on an anti-CA125 antibody immobilized on microtitration wells. Serum samples from high-grade serous EOC or patients with varying benign conditions and healthy controls were analyzed.

Results: CA125MGL specifically recognized the CA125 isoform produced by EOC, whereas the recognition of CA125 from nonmalignant conditions was reduced. Serum CA125MGL measurement discriminated better patients with EOC from benign conditions compared with conventional immunoassay. Importantly, discrimination was particularly improved for marginally increased CA125 values that are common in benign conditions. Our data also showed earlier detection of EOC progression, suggesting that the CA125MGL also has potential to improve earlier diagnosis of EOC.

Conclusions: The CA125MGL concept could help reduce the false-positive rates of conventional CA125 immunoassays. It carries further potential to improve early diagnosis of EOC as well as follow-up of treatment response. Finally, using appropriate combinations of lectins and antibodies, applying the test concept presented here can also be explored for other diagnostic targets, where changes in glycosylation are indicative of an ongoing disease process.

Citation Format: Kaisa Huhtinen, Kamlesh Gidwani, Henna Kekki, Johanna Hynninen, Liina Salminen, Antti Perheentupa, Matti Poutanen, Annika Auranen, Seija Grenman, Urpo Lamminmäki, Olli Carpén, Kim Pettersson. A cancer-specific detection of serum CA125 improves differential diagnosis of epithelial ovarian cancer from benign conditions. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B15.
Abstract B37: Prevention of epithelial ovarian cancer by eliciting P4/RIPK1-dependent necroptosis
Authors: Bitler Benjamin , Yamamoto Tomomi , Bales Elise , Watson Zachary , Behbakht Kian , Johnson Joshua
Published: August 2018

High-grade serous epithelial ovarian cancer (HGSOC) is the deadliest gynecologic disease, commonly diagnosed in peri/postmenopausal women. Annually, HGSOC accounts for over 140,000 deaths worldwide. Because we lack effective early detection methods, over 75% of HGSOC are diagnosed at stages III/IV, with abysmal 5-year survival rates of 37/25%, respectively. Recently, progesterone (P4) was observed to promote the elimination of p53-null cells from the fallopian tube by inducing necroptosis, a regulatable form of non-apoptotic cell death. Activation of necroptosis depends on activation of receptor-interacting kinase proteins 1 and 3 (RIPK1 and RIPK3). Several additional factors suggest that P4 may have a key protective role versus HGSOC; protection versus tumorigenesis is reduced at the time of menopause due to decreasing P4 levels. Several studies examining the use of a synthetic progesterone (medroxyprogesterone acetate, MPA) observed decreases in the incidence of HGSOC. Also, MPA had a significant clinical benefit in 20-40% of pretreated ovarian cancer patients. Further, 75% of HGSOC have decreased expression of progesterone receptor (PGR). With lower P4 at menopause, p53 mutated fallopian tube epithelial cells may therefore escape necroptosis and progress to HGSOC. All of the above information led to our hypothesis that activating P4 signaling will induce necroptosis in HGSOC tumor cells.

Examining The Cancer Genome Atlas (TCGA) database for regulators of necroptosis, we found that increased expression of the P4 receptor (PGR), necroptosis effectors TNFα, and RIPK1/3 corresponded to significantly higher overall survival in 484 HGSOC cases. Using OVSAHO (BRCA2-deleted) cells, we show that P4 and/or TNFα treatment increases TNFα, RIPK1, and RIPK3 expression. In OVSAHO cells, we observed a significant decrease in cell number following treatment with P4 and/or TNFα. Also, we confirmed that P4 treatment was able to induce a necroptotic response distinct from classical apoptosis. Upon examination of normal fallopian tube tissue, borderline tumors, and advanced HGSOC, we detected expression of necroptosis effectors RIPK1 and 3, suggesting an intact necroptosis pathway. These observations point towards an HGSOC prevention strategy that favors the elimination of “founder” cells by activating necroptosis downstream of P4. These data may be particularly useful for the management of BRCA1/2 mutant patients, as these women experience menopause significantly earlier than noncarriers.

Citation Format: Benjamin Bitler, Tomomi Yamamoto, Elise Bales, Zachary Watson, Kian Behbakht, Joshua Johnson. Prevention of epithelial ovarian cancer by eliciting P4/RIPK1-dependent necroptosis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B37.
Abstract B57: Adhesion and beyond: CD44 in ovarian cancer spheroids
Authors: Sacks Joelle D. , Main Hilal Gurler , Muralidhar Goda G. , Elfituri Osama , Xu Hao-Liang , Kajdacsy-Balla Andre A. , Barbolina Maria V.
Published: August 2018

Epithelial ovarian carcinoma (EOC) metastasis is characterized by the shedding of malignant cells from the surface of the primary tumor and their implantation onto the peritoneal surface lining the abdominal cavity in addition to more distant sites. This shedding of malignant cells from the primary site results as either single cells or free-floating multicellular aggregates, known as spheroids, in the ascites. Although single cells and spheroids may potentially seed metastases, considerable evidence now suggests that the aggregation of cells is important for anchorage-independent cell survival and growth, and spheroid formation may represent an important intermediate survival mechanism to facilitate EOC dissemination. Recent studies have uncovered human transmembrane cell adhesion molecule CD44 standard (CD44s) isoform expression correlating with high-grade and advanced-stage ovarian carcinoma. Moreover, it has also been suggested that CD44s may be an important mediator of ovarian cancer cell implantation in the peritoneal cavity, enhancing the metastatic potential of ovarian cancer cells. Thus, we investigated the functional significance of CD44 standard by means of in vitro culture experiments with ovarian cancer spheroids. After characterizing 8 EOC cell lines, a spectrum of CD44 expression was observed and two cell lines with high CD44s (ES-2 and SKOV-3) and two cell lines low in CD44s (OVCAR-4 and OVSAHO) were chosen for further testing. Interestingly, the high CD44s-expressing cell lines formed larger spheroids that adhered significantly faster to a monolayer of primary mesothelial cells. Stable knockdown of CD44 using CRISPR/Cas9 in these 2 cell lines reduced spheroid formation, suggesting that differential expression of CD44 standard plays a role in cell-cell adhesion. When ES-2 CD44-/- cells were injected i.p. into athymic nude mice, decreased ascites production and mesentery tumor burden were observed as well as increased overall survival. However, CD44 knockdown also significantly increased metastasis to the lung, suggesting a suppressive role for CD44 in EOC metastasis as well. These in vivo data imply that CD44 enhances the metastatic potential in ovarian cancer spheroids influencing peritoneal tumor growth while suppressing metastasis to lung tissue.

Citation Format: Joelle D. Sacks, Hilal Gurler Main, Goda G. Muralidhar, Osama Elfituri, Hao-Liang Xu, Andre A. Kajdacsy-Balla, Maria V. Barbolina. Adhesion and beyond: CD44 in ovarian cancer spheroids. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B57.
Abstract IA23: How does the immune system contend with intratumoral heterogeneity in ovarian cancer?
Authors: Nelson Brad H.
Published: August 2018

The presence of tumor-infiltrating T cells and B cells is associated with markedly prolonged survival in high-grade serous ovarian cancer (HGSC), yet we have only a rudimentary understanding of how the immune system contends with the continually evolving tumor genome. HGSC exhibits extensive spatial heterogeneity at diagnosis, and tumors undergo dramatic changes in response to treatment. In long-term survivors, the immune system presumably deploys mechanisms that successfully respond to these changes. With better understanding, it may be possible to exploit these mechanisms to create more effective immunotherapies.

We have shown that the most prognostically favorable immune responses involve both cytolytic and antibody-based mechanisms, suggesting important cooperative interactions between the T cell and B cell branches of the immune system. By studying serial tumor samples, we have uncovered extensive temporal dynamics of the antitumor immune response. We found that neoadjuvant chemotherapy can enhance preexisting immune responses yet generally fails to initiate responses in tumors that lack immune infiltrates at baseline. Indeed, with better understanding, these immunologically inert or “cold” tumors may represent an attractive therapeutic opportunity, as they can express high levels of tumor-specific antigens with corresponding systemic T-cell and antibody responses.

Currently, we are looking at the issue of spatial heterogeneity by subjecting tumor samples collected from different anatomical locations to comprehensive genomic and immunologic analyses, including mutational profiling, RNA-seq, TCR/BCR-seq, and multi-plexed immunohistochemistry. This work is providing novel insights into the relationship between the clonal architecture of tumors and antitumor immunity. Our findings to date indicate that tumor deposits with a high degree of clonal heterogeneity generally have low densities of immune infiltrates, suggesting a means by which tumor evolutionary processes are insulated from immunologic attack. Furthermore, TCR-seq experiments are providing early evidence that T-cell clones track with individual tumor clones across space, suggesting that the immune system contends with intratumoral heterogeneity by battling each tumor clone individually. The presentation will address the implications of these findings on the design of more effective immunotherapies for HGSC and related cancers.

Citation Format: Brad H. Nelson. How does the immune system contend with intratumoral heterogeneity in ovarian cancer? [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA23.
Abstract A30: Preclinical study of a combination of mocetinostat (HDAC inhibitor) and 5-AZA-dC (decitabine) in chondrosarcoma
Authors: Sheikh Tahir N. , Patwardhan Parag , Schwartz Gary K.
Published: August 2018

Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Previous reports have shown that the interaction of DNA methylation and histone modification regulates gene expression, and targeting these mechanisms could be a potentially novel approach to treat cancer. In the present study, we hypothesized that concurrent inhibition of histone acetylation and DNA methylation could result in decreased viability of chondrosarcoma cells. To test this, we used a panel of chondrosarcoma cell lines including IDH wild type (CH2879), IDH1 mutant (JJ012), and IDH2 mutant (SW1353) cell line. Results from our in vitro proliferation assay showed that combination of sub-IC50 concentrations of the DNA methyltransferase (DNMT) inhibitor decitabine (5-AZA-dC) and a spectrum-selective class I and IV histone deacetylase (HDAC) inhibitor mocetinostat (MGCD0103) resulted in decreased cell viability, which appears to be at least additive, in all the three chondrosarcoma cell lines when compared to either of the two drugs alone. Western blot analysis showed induction of cleaved Poly-ADP Ribose Polymerase (PARP) as well as cleaved caspase 3, known markers of apoptosis. Consistent with augmented DNA damage, combination of decitabine and MG103 markedly increased the levels of phospho-H2AX, a DNA damage marker, and proapoptotic BH3-only proteins such as Bim. Combination treatment also resulted in increased induction of histone acetylation (AcH3 and AcH4) and increased expression of E-cadherin, an important tumor suppressor gene. Cell cycle analysis showed induction of sub-G1 population phase in combination treatment. Previous reports have shown that CpG methylation is a critical event in transcriptional repression of hypermethylated genes in cancer. To this end, we show that combination treatment with mocetinostat and 5-AZA-dC results in downregulation of (H3K9) methylation. Taken together, our data strongly suggest that combination treatment with mocetinostat and decitabine is a novel treatment approach and merits evaluation in the treatment of chondrosarcoma.

Citation Format: Tahir N. Sheikh, Parag Patwardhan, Gary K. Schwartz. Preclinical study of a combination of mocetinostat (HDAC inhibitor) and 5-AZA-dC (decitabine) in chondrosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A30.
Abstract B19: Tumor-suppressive activities associated to MIR10A-5p expression in developmental sarcomas
Authors: Herrero-Martin David , Rello-Varona Santiago , Garcia-Monclus Silvia , Huertas-Martinez Juan , Almacellas-Rabaiget Olga , Helman Lee J. , Tirado Oscar M.
Published: August 2018

Ewing sarcoma (ES) and alveolar rhabdomyosarcoma (ARMS) are pediatric sarcomas characterized by tumor-specific translocations. Besides acting as a direct modulator of transcription, fusion proteins appear to exert its oncogenic functions by epigenetic modifications on the transcriptome. Therefore, the identification of specific DNA methylation markers would be helpful for understanding their pathogenetic mechanism as well as for developing new therapeutic strategies. By using the Illumina Infinium HumanMethylation450 we have analyzed the methylome of ES and ARMS tumors and cell lines. ES and ARMS samples showed, among other epigenetic alterations, differential hypermethylation in the promoter of MIR10A-5p, thus suggesting an inhibition of its expression. Expression of MIR10A-5p is effectively low in ES and ARMS cell lines and patient samples and the treatment with the epigenetic modifier 5-aza restores its expression. MIR10A-5p stable overexpression in two ES (A673 and TC252) and two ARMS (Rh4 and RMS13) cell lines reduces proliferation, diminishes clonogenic growth, and decreases cellular migration. Cell death induced by MIR10A-5p reintroduction is only achieved in the p53wt ES cell line TC252. Proteomic profile of A673 MIR10A-5p cells is altered as assessed by iTRAQ. Protein kinase CRKL is one of the proteins downregulated due to MIR10A-5p reintroduction. Indeed, CRKL expression is also reduced in the other three MIR10A-5p stable overexpression models. Doxycycline-induced silencing of CRKL in ARMS cell line Rh30 provokes cell growth reduction in vitro and in vivo, G0/G1 arrest, and a decrease in the clonogenic capacity (Yeung CL et al., 2013). Interestingly, the migratory ability of Rh30 and A673 CRKL-silenced cells is also impaired. Thus, our results uncover MIR10A-5p as a putative tumor suppressor in ES and ARMS, suggesting a link between MIR10A-5p and CRKL. Further characterization of the oncogenic role of CRKL and the molecular mechanisms associated to MIR10A-5p tumor-suppressor activities in ES and ARMS is ongoing.

Citation Format: David Herrero-Martin, Santiago Rello-Varona, Silvia Garcia-Monclus, Juan Huertas-Martinez, Olga Almacellas-Rabaiget, Lee J. Helman, Oscar M. Tirado. Tumor-suppressive activities associated to MIR10A-5p expression in developmental sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B19.
Abstract IA08: Analysis of a histiocytic carcinoma in the dog: The canine's utility in fetching disease genes
Authors: Parker Heidi G. , Cadieu Edouard , Shearin Abigail , Rutteman Gerard , Ostrander Elaine A.
Published: August 2018

Histiocytic sarcoma (HS) is a rare human hematopoietic neoplasm that accounts for less than 1% of hematolymphoid cancers and occurs either concurrently with or subsequently to B- or T-lymphoblastic lymphoma and other B-cell cancers. As such, its genetic etiology has been difficult to establish. HS in dogs, by comparison, shows strong breed specificity with 20% of flat-coated retrievers and 25% of Bernese mountain dogs (BMD) estimated to develop the disease in their lifetime. These two affected breeds share no recent common ancestors and display differences in disease presentation, offering a unique system in which to investigate the genetic predisposition and progression of HS. Our lab has previously undertaken genome wide association study (GWAS) on both BMD and FCR collected in North America and Europe and identified four significantly associated loci, two in BMD and two in FCR. In each breed, only one locus was segregating with disease in the American population while both were present in the European populations. There were no loci in common between the two breeds. Fine mapping of the common BMD HS locus on canine chromosome (CFA) CFA11 revealed a single haplotype spanning the MTAP (methylthioadenosine phosphorylase) gene and part of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, which was present in 96% of BMD with HS. The European BMD also showed a significant locus on CFA14 that may be approaching fixation in the American dogs based on allele frequency calculations. We have done targeted resequencing of the CFA14 locus in a subset of European BMD and are in the process of filtering the identified mutations. Promising candidate genes in this region include GRM8, which inhibits the cyclic AMP cascade in the central nervous system and has shown to be involved in several of cancers; POT1, which is involved in telomere length maintenance and protection; and HYAL4, a hyaluronidase potentially involved in extracellular matrix degradation. In addition, the laboratory has generated 30x sequence from two HS cases and a control BMD, two FCR cases, and a control and low pass sequence from 16 dogs (8 cases and controls) in order to identify the likely causative mutations in each of the associated loci as well as breed specific mutations that may predispose the dogs to aggressive cancers.

Citation Format: Heidi G. Parker, Edouard Cadieu, Abigail Shearin, Gerard Rutteman, Elaine A. Ostrander. Analysis of a histiocytic carcinoma in the dog: The canine's utility in fetching disease genes [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA08.
Abstract IA15: Using gene expression analyses to identify MPNST therapeutics
Authors: Ratner Nancy
Published: August 2018

Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas that are a major source of mortality in neurofibromatosis type 1 (NF1) patients. In NF1 patients, these devastating sarcomas often develop in existing benign neurofibroma. We hypothesized that some over-expressed genes in solid tumors would be driver genes and/or mark critical pathways and therapeutic targets. In one approach to identify MPNST driver genes we targeted 130 genes up-regulated in neurofibroma and MPNSTs versus normal cells in a lentiviral short hairpin (sh) RNA screen. A counter-screen in RAS mutant cells excluded common RAS-pathway driven genes. We identified 7 genes specific for survival of MPSNT cells, including the transcription factor MEIS1. In a second approach, we targeted known oncogenes that were expressed at higher levels in MPNST than in normal cells, including Aurora Kinase A. MPNST responded to the Aurora A kinase inhibitor alisertib, and the combination of alisertib and HSV1716, a virus derived from HSV-1 and attenuated by deletion of RL1, exhibited significantly increased antitumor efficacy compared to either monotherapy. Supported by NIH-NS-084855 (N.R. and Largaespada, D.), NS-28840 (N.R.), and NS-086219 (N.R. and Cripe, T.P.).

Citation Format: Nancy Ratner. Using gene expression analyses to identify MPNST therapeutics [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA15.
Abstract IA17: Pathway dependence and drug synergism in NF1-associated malignant peripheral nerve sheath tumors using the zebrafish model
Authors: Ki Dong Hyuk , He Shuning , Look A. Thomas
Published: August 2018

Malignant peripheral nerve sheath tumors (MPNSTs) are very aggressive and often metastatic soft tissue sarcomas, which are frequently found in patients who have neurofibromatosis type 1 (NF1). Currently, surgical excision is the only curative therapy for MPNST; however, many patients have unresectable or metastatic tumors at diagnosis and the recurrence rate after surgery is high. Chemotherapy regimens are only partially effective and associated with significant toxicity that can severely reduce quality of life. To establish a preclinical model of this highly malignant tumor, we developed a faithful in vivo MPNST transplantation assay to assess pathway dependence. mCherry-labeled MPNSTs cells were harvested from nf1 and p53 mutant zebrafish and implanted into the pericardial space of embryos at 2 dpf. 24 hours later, we added multiple pathway inhibitors or DMSO vehicle to the fish water and the embryos were incubated for 4 days. Drugs in clinical use for MPNST were tested such as inhibitors of MEK (trametinib), RTK (sunitinib), mTOR (rapamycin), HSP90 (ganetespib), microtubules (vincristine), alkylating agents (cytoxan), XPO I (selinexor), topo II (etoposide), and topo I (topotecan). Of these, only topotecan and selinexor showed significant cytotoxic responses at their MTDs. These two inhibitors were also active in human MPNST cell lines, and we are testing to see if their tumor cell cytotoxic effects are synergistic. Clinical trials of Topo I and XPO I inhibitors have not been reported for MPNST, and our results indicate that these two drugs should be evaluated clinically in patients with this tumor. We are now assessing FDA-approved drugs to identify those that synergize with topotecan and selinexor to induce sustained MPNST cell death and improved survival at tolerable dosages. Our long-range goal is to define active combinations of three or more non-cross resistant drugs that exhibit synergistic and sustained tumor cell cytotoxicity at dosages tolerable to the host.

Citation Format: Dong Hyuk Ki, Shuning He, A. Thomas Look. Pathway dependence and drug synergism in NF1-associated malignant peripheral nerve sheath tumors using the zebrafish model [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA17.
Abstract PR12: Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape
Authors: Lu Chao , Jain Siddhant , Hoelper Dominik , Bechet Denise , Ran Leili , Andrulis Irene , Dickson Brendan , Majewski Jacek , Thompson Craig , Chi Ping , Garcia Benjamin , Jabado Nada , Lewis Peter , Allis C. David
Published: August 2018

Several types of bone/cartilage tumors reportedly contain high-frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. We find that the H3 lysine 36 to methionine (H3K36M) mutation, identified in >90% of chondroblastomas, impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases, resulting in globally diminished H3K36 methylation. Depletion of H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy H3K36M mutation. Following the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of polycomb target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas where novel K36M/I mutations in H3.1 are identified in rare pediatric cases.

This abstract is also being presented as Poster A12.

Citation Format: Chao Lu, Siddhant Jain, Dominik Hoelper, Denise Bechet, Leili Ran, Irene Andrulis, Brendan Dickson, Jacek Majewski, Craig Thompson, Ping Chi, Benjamin Garcia, Nada Jabado, Peter Lewis, C. David Allis. Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR12.