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Background: Radiation therapy (RT) is a mainstay of treatment for most women with breast cancer (BC). Despite this treatment, response remains heterogenous for women with estrogen receptor positive (ER+) BC. Thus, approaches that result in radiosensitization of aggressive ER+ disease are critically needed. We performed a radiosensitizer screen paired with transcriptomic and proteomic data from ER+ models treated +/-RT to identify potential mediators of RT resistance. Methods: Clonogenic survival assays were used to determine RT sensitivity of 21 BCC lines as well as radiosensitization with drug treatment. IC50 values were determined for 130 clinical compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Microarray and RPPA data was used for differential gene/protein expression and pathway analysis. AlamarBlue was used to determine IC50 values of the MDM2 inhibitor AMG-232. Western blot analysis of Cleaved PARP was used to measure apoptosis and Cyclins A and E to measure cell cycle progression. Results: Our radiosensitizer screen nominated the MDM2 inhibitor (JNJ-26854165) as one of the most effective drugs in treating RT-resistant BC cell lines (R2= 0.43, p-value 10uM). Clonogenic survival assays demonstrated that MDM2 inhibition at sub-IC50 doses leads to radiosensitization in p53 WT ER+ cell lines (MCF-7 rER: 1.37-1.66;ZR751 rER: 1.30-1.65). In contrast, p53 MT ER+ cells did not demonstrate significant radiosensitization (T47D rER: 0.94-1.11). Combination of AMG-232 and RT led to an increase in apoptosis compared to RT alone in ER+ p53 WT cells but not p53 MT cells. Additionally, combination treatment led to differential cylin expression in p53 WT cells but not p53 MT cells. In vivo studies testing MDM2 inhibition with RT in p53 WT and MT orthotopic and PDX models are ongoing. Conclusions: Our novel radiosensitizer screen identifies MDM2 as a potential mediator of radioresistance in ER+ BC. Additionally, MDM2 inhibition confers radiosensitization in a p53 dependent manner in ER+ BC and may represent a tractable clinical strategy in women with p53 WT BC. Citation Format: Cassandra L. Ritter, Benjamin C. Chandler, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Amanda Zhang, Tanner Ward, Mattia Cremona, Bryan Hennessy, Lori J. Pierce, Corey W. Speers. A radiosensitizer screen identifies a novel role for MDM2 inhibition in the radiosensitization of ER+ breast cancers in a p53 dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6270.

Purpose: Brain metastases of breast cancer cause neurocognitive damage and are incurable. We evaluated in experimental brain metastasis model a role of temozolomide, an oral brain permeable alkylating agent characterized by significant uptake in the central nervous system, in the prevention of brain metastases of breast cancer. Material and methods: To assess preventive role of temozolomide, mice were inoculated with 175,000 triple-negative 231-BR-EGFP cells in 0.1 mL PBS in the left ventricle of the heart. Three days after tumor cell inoculation, mice were randomized to temozolomide at the dose of 50 mg/kg delivered by oral gavage in saline, 5 days a week for 4 weeks, or vehicle. Subsequent experiments used temozolomide doses of 25, 10, 5, 1 and 0.5 mg/kg. To evaluate the efficacy of temozolomide in treating established BM, mice received temozolomide (50 mg/kg) beginning on either day 18 or day 24 post-injection of 231-BR-EGFR cells, 5 days a week for two and one week, respectively. To investigate the impact of temozolomide on survival, mice injected with 231-BR-EGFP cells were randomized to vehicle, temozolomide on days 3-14, or temozolomide on days 17-28 post-injection, per the schedule described above. To determine the functional contribution of MGMT expression in the BM preventive model, similar experiments were performed using 231-BR-EGFP cells with induced MGMT expression, and MGMT-positive Jimt-1 cells. Metastases were counted in step sections of one hemisphere of each brain. Additionally, the percentage of MGMT-positive tumor cells in 62 patient-matched sets of breast cancer primary tumors and resected brain metastases was determined immunohistochemically. Results: Temozolomide, when dosed at 50, 25, 10 or 5 mg/kg, 5 days/week, beginning 3 days after inoculation, completely prevented the formation of experimental brain metastases from MGMT-negative 231-BR-EGFP cell line. At a 1 mg/kg dose, temozolomide prevented 68% of large brain metastases, and was ineffective at a dose of 0.5 mg/kg. When the 50 mg/kg dose was administered beginning on days 18 or 24, temozolomide efficacy was reduced or absent. Both schedules of temozolomide (days 3-14 and days 17-28) significantly increased survival (P = .0003 by long-rank test). Earlier administration of temozolomide resulted in long term survival of 6 and 2 out of 10 mice, respectively; a significant difference compared to vehicle (P < .0001 and .0003, respectively).Temozolomide was ineffective at preventing brain metastases in the MGMT-positive 231-BR-EGFP and Jimt-BR3 sublines. In 62 patient-matched sets of primary breast tumors and resected brain metastases 43.5% of the specimens had concordant low MGMT expression, while in another 14.5% sets high MGMT staining in the primary tumor corresponded with low staining in the brain metastasis. Conclusions: Temozolomide profoundly prevents the outgrowth of experimental brain metastases of breast cancer in a MGMT-dependent manner. The majority of patients had low MGMT expressing brain metastases. These data provide a compelling rationale for investigating preventive efficacy of temozolomide in high-risk advanced breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-11-04.

Post-translational modifications (PTMs) of histone tails can function as epigenomic regulators of the cellular response to DNA damage. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone PTM involved in the DNA damage response. H2Bub1 localizes to double-strand breaks where it functions to decondense chromatin, making it accessible to DNA repair factors. H2Bub1 is also known to be enriched at coding sequences of highly expressed genes, demonstrating the fundamental importance of H2Bub1 for an open chromatin structure that facilitates transcription. H2Bub1 is a dynamic process, written predominantly by the E3 ubiquitin ligase complex of the ring finger proteins RNF20 and RNF40 and erased by multiple deubiquitinases, including those from the ubiquitin-specific peptidase subfamily USP7 and USP44. RNF20 is reported to interact with p53. Platinum drugs are used as standard-of-care for the treatment of high-grade serous ovarian cancer (HGSOC) based on their capacity to damage DNA. We sought to determine the response of ubiquitinated histone tails to a platinum drug with the goal of improving the efficacy of current best practice therapy for HGSOC. Following treatment of a wild-type p53 cell line with an IC75 dose of cisplatin, we combined H2Bub1 chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to determine genomic enrichment of H2Bub1 in response to DNA damage. Further, we coupled this with RNA-seq (RNA sequencing) to enable correlation of genomic enrichment of H2Bub1 with levels of gene expression. KEGG pathway and gene ontology analyses demonstrated greatest H2Bub1 enrichment in response to DNA damage in p53 signalling pathway genes, including CDKN1A, BBC3, MDM2 and BAX. This correlated with increased expression of p53 target genes in response to DNA damage. Wild-type TP53 itself did not show H2Bub1 enrichment upon treatment with cisplatin. H2Bub1 ChIP experiments were repeated in HGSOC mutant p53 cell line models, specifically OVCAR-3 with the known gain-of-function TP53 mutation R248Q and Kuramochi with the missense TP53 mutation D281Y. H2Bub1 was not enriched in response to DNA damage at wild-type p53 gene targets in these cell lines, nor were these genes highly expressed. We therefore propose that DNA damage induced H2Bub1-enrichment in mutant p53 cell lines will have an entirely distinct genomic profile compared to wild-type p53 cell lines, with important roles in enabling the expression of mutant p53 target genes in HGSOC. Experiments are currently under way to directly address this hypothesis. Lastly, we have used clonogenic cell survival assays to demonstrate that downregulation of RNF20, either transiently using short interfering (si) RNA or stably,using short hairpin (sh) RNA, decreased the ability of HGSOC cells to survive treatment with cisplatin. Down-regulation of H2Bub1 by downregulation of its E3 ubiquitin ligase RNF20 may represent a novel treatment strategy to increase the efficacy of DNA damage-based therapy for HGSOC. Citation Format: Alexander J. Cole, Kristie-Ann Dickson, Christopher Liddle, Clare Stirzaker, Roderick Clifton-Bligh, Deborah J. Marsh. Cisplatin-induced DNA damage modifies the chromatin landscape of histone H2B monoubiquitination in a p53-dependent manner. [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 A06.

Aberrant hedgehog signaling has been implicated in many human cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. In fact, 30% of MB tumors are driven by aberrant activity of the Sonic hedgehog (SHH) signaling pathway. Our efforts focus on unraveling the downstream components of this pathway to better understand medulloblastoma and better inform therapeutic treatment development and decisions. While some of the signaling alterations in SHH MB are due to gene mutations or amplifications, others may have their roots in epigenetics. Therefore, we examined expression levels of several candidate epigenetic regulators in our systems and identified lymphoid-specific helicase (HELLS) as a gene whose expression is markedly induced by SHH. HELLS is a unique member of the SNF2 family of chromatin remodelers with multiple epigenetic functions in DNA methylation, histone acetylation and methylation, and chromatin remodeling. Additional roles in transcription activation and DNA repair have also been reported. Of interest, HELLS was shown to delay senescence by inhibiting the expression of CDKN2A, the genetic locus of P16INK4A, a key tumor suppressor whose inactivation has recently been reported as critical to MB progression. Confirming our initial finding, we observed considerably higher levels of HELLS in primary cultures of cerebellar granule neuron precursors (CGNPs) treated with SHH and in SmoA1 mouse MB tumor tissue when compared to adjacent normal cerebellum. Our preliminary analysis of a large cohort of MB patients also indicates overexpression of HELLS in human SHH MB. Bioinformatic promoter analysis and experiments with SHH pathway inhibitors suggest regulation of HELLS expression through members of the SHH proliferation program. Downstream effectors of SHH include GLI1/2 and YAP1. Inhibition of GLI1 and GLI2 with Gant61 in both CGNPs and cultured mouse MB cells resulted in a reduction of HELLS, but an increase of apoptosis markers may indicate this reduction is due to cell death rather than specific downregulation of HELLS. In contrast, verteporfin, an inhibitor of the interaction between the transcriptional co-activator YAP1 and the transcription factor TEAD1, resulted in a reduction of HELLS at the mRNA and protein level without a concomitant increase of apoptosis markers. These results suggest that transcriptional upregulation of HELLS in mouse cerebellar progenitors and MB tumors is mediated by the SHH effector YAP1. Experiments to confirm direct involvement of YAP1 in HELLS gene transcription are underway. Future studies to understand the role of HELLS in SHH MB have the potential to provide a better understanding of the disease and new avenues for development of targeted treatments to improve the lives of these patients. Citation Format: M. Hope Robinson, Anna M. Kenney. HELLS is upregulated in Sonic hedgehog-associated medulloblastoma and proliferating cerebellar progenitors in a YAP-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4134.

Deregulated expression of MYC family oncogenes MYC, MYCN and MYCL (here collectively referred to as MYC) occurs in many types of human tumors, and is often associated with aggressive tumor development and poor prognosis. In mouse tumor models, inactivation of MYC often leads to tumor regression with well-tolerated side effects, suggesting that MYC is a potential and suitable target for anti-cancer therapy. At present there are no drugs targeting MYC in the clinic, and transcription factors like MYC are considered difficult to target. However, MYC function is strictly dependent on interaction with cofactors, and targeting such interaction may be a plausible way to combat Myc. We have used cell-based screens utilizing Bimolecular Fluorescence Complementation (BiFC) and split Gaussia luciferase (G-Luc) to identify small molecule inhibitors of the interaction between MYC and its essential partner MAX. Several potent MYC:MAX interaction inhibitors have been identified in these screens and have been validated by other protein-protein interactions (PPI) assays such as in situ Proximity Ligation Assay (isPLA), Fluorescence Resonance Energy Transfer (FRET), Surface Plasmon Resonance (SPR) and coimmunoprecipitations. In general the identified molecules fall into two categories: those that inhibit MYC:MAX interactions not only in cells but also in vitro using purified MYC and MAX, and those that inhibit the interaction in cells but not in vitro, suggesting an indirect mode of action. Molecules of both categories inhibit cell growth and viability of a variety of tumor cells in culture with high efficacy in a MYC-dependent manner. So far, one of the molecules have been utilized for cancer treatment in mouse tumor models and found to significantly inhibit tumor growth and improve survival. These molecules have the potential to become important tools in the studies of MYC function in cells and in vivo as well as potentially basis for drug development for treatment of MYC-driven tumors. We have thus provided proof of principle that our screening systems are able to identify potent PPI inhibitors and we are at present setting up similar systems to screen for inhibitors of other MYC:cofactor interactions of relevance for specific tumor types. Citation Format: Alina Castell, Karin Ridderstråle, Qinzi Yan, Fan Zhang, Per Hydbring, Marcela Franco, Jacob Goodwin, Inga Müller, Siti Mariam Zakaria, Lars Johansson, Lars-Gunnar Larsson. New small molecules targeting MYC:MAX interactions that inhibits tumor cell growth in a MYC-dependent manner. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1227.

Adenocarcinoma of the proximal stomach is the fastest rising malignancy in North America, and is associated with a high rate of recurrence to the peritoneum. As part of cancer treatment, the majority of patients undergo at least one invasive surgical procedure. Recent clinical data has linked postoperative infection complications with adverse oncologic outcomes; however, the underlying mechanisms are unclear. Emerging evidence suggests that the release of TNFa, a key inflammatory cytokine during infection facilitates cancer progression. The role and mechanisms the gram-negative bacterial infections play in facilitating the metastatic potential of gastric cancer to the peritoneum is entirely unknown. We hypothesized that incubation of gastric cancer cells and MC with heat-inactivated E. Coli or LPS can augment gastric cancer cell adhesion and invasion via TNFR1 signaling and increase the potential of peritoneal metastasis. Incubation of human gastric cancer cells or/and MC with heat inactivated E. coli, LPS or TNFa significantly increased in vitro adhesion 3-4 folds to MC and enhanced in vitro invasion. These enhanced cell adhesion and invasion phenotypes following incubation with LPS or E. coli were attenuated at three levels: inhibition of TLR4 (Eritoran), inhibition of TNFR1 (anti-TNFR1/isotype control antibodies) or p38 MAPK inhibitor (BIRB0796). TNFa treatment also increases CD54, CD44 and CD29 expression on cancer cells and MC. To further validate in vitro results, a novel ex-vivo murine peritoneal metastasis model is developed. We report that ex vivo gastric cancer cells adhesion to murine peritoneum is augmented by overnight LPS, E. Coli and TNFa treatments and this effect was abrogated when TNFR1-/- mouse peritoneum is used or in the presence of TNFR1 monoclonal or isotype control antibodies. These findings implicate TNFa in the process of gastric cancer metastasis to peritoneum in the context of systemic infection and identify TNFa as potential therapeutic targets. Citation Format: Malak Alzahrani, Betty Giannias, France Bourdeau, Rafael Kayano, Lorenzo Ferri. Gram-negative bacterial infection enhances the potential of gastric adenocarcinoma peritoneal metastasis via TNFR1 dependent manner. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A29.

Tumor microenvironment (TME) plays an important role in cancer progression; however, the communication between TME and cancer cells remains largely unclear. CD44, a type I cell surface receptor functioning in cell-cell and cell-extracellular matrix interactions, has been implicated in tumor formation and progression. Using gain-of-function and loss-of-function approaches, we report in this study that serglycin (SRGN), a glycoprotein secreted from cancer-associated fibroblasts (CAFs) as well as aggressive cancer cells, works to promote the malignant behaviors of non-small cell lung cancer cells through its interaction with CD44. We showed that SRGN promotes cell transformation and tumor formation and metastasis using in vitro and in vivo tumorigenic assays. Furthermore, we showed that SRGN induced lung cancer cell stemness in a CD44-dependent manner. SRGN enhanced cancer cells sphere formation via Nanog induction, accompanied with increased drug resistance and resistance to anoikis. We also demonstrated that SRGN promotes cell migratory activity through promoting epithelial-mesenchymal transition (EMT) and enhancing adhesions turnover by upregulating vimentin expression and SRC activity via CD44/NF-KB/Claudin-1 axis. In support, Claudin-1 expression was tightly associated with SRGN expression in primary lung cancer tissues (p < 0.0001). In summary, we demonstrate that the crosstalk between tumor cells and TME promotes malignant phenotypes through the interaction of tumor-borne CD44 and CAF- or tumor-derived SRGN, suggesting that targeting CD44 or its ligands may represent an attractive strategy for development of cancer therapy. Citation Format: Jing-You Guo, Shiaw-Wei Tyan, Han-Shui Hsu, Fen-Yau Li, Jeou-Yuan Chen. Secreted serglycin in tumor microenvironment promotes tumor malignancies in a CD44-dependent manner. [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 1541. doi:10.1158/1538-7445.AM2015-1541

Inflammatory breast cancer (IBC) is a highly metastatic, aggressive, and fatal form of breast cancer that accounts for about 1 to 5% of all breast cancers diagnosed in the United States. At present, the molecular mechanisms that underlie the aggressive phenotype of IBC are not known. Interferon induced transmembrane protein1 (IFITM1) is a member of interferon stimulated genes (ISGs), whose overexpression has been linked to several malignancies, but its role in inflammatory breast cancer is not known. The primary role of IFITM1 is to stop the spread of viral pathogens in the host's cells, but its constitutive overexpression has been shown to enhance malignancy. IFITM1 expression is known to be regulated by type 1 interferon through activation of the canonical JAK-STAT pathway; however, recent studies suggest that non-canonical signaling pathways involving STAT2 homodimers can also regulate IFITM1 expression. In the present study, we determined whether IFITM1 plays a critical role in driving the aggressive phenotype of three IBC cell lines; SUM149; MDA-IBC-3, and SUM190. Western blot and RT-PCR analyses indicated that IFITM1 was constitutively overexpressed at the protein (∼5- to 10-fold) and mRNA (∼4-fold) level, respectively, in SUM149 and MDA-IBC-3 cells but not expressed in non-IBC MCF-7 luminal breast cancer cells, and that its overexpression strongly correlated with increased proliferation, migration and invasion, and enhanced colony formation. Notably, the aggressive phenotype of SUM149 and MDA-IBC-3 cells was completely reversed by siRNA knockdown of IFITM1. Specifically, we found that suppression of IFITM1 significantly reduced the ability of SUM149 and MDA-IBC-3 cells to proliferate, migrate and invade, and form colonies. Furthermore, we found that IFITM1 expression and promoter activity were completely abolished due to STAT2 knockdown and that BRG1 (Brahma-related-gene 1) was crucial for the expression of IFITM1 and STAT2 in the IBC cells. Moreover, we found that knockdown of BRG1; a member of the mammalian chromatin-remodeling complex markedly reduced IFITM1 and STAT2 proteins, but not STAT1, in SUM149 and MDA-IBC-3 cells, thus supporting a critical role for STAT2/BRG1 in the regulation of IFITM1 expression in these cells. Taken together, this data suggests that constitutive overexpression of IFITM1 in inflammatory breast cancer cells enhances the aggressive phenotype of these cells, and that STAT2 and BRG1 play pivotal roles in driving IFITM1 overexpression. Additionally, this data suggests that IFITM1 may be a suitable candidate as a novel therapeutic target and a potential prognostic maker for inflammatory breast cancer. Citation Format: Joshua Were Ogony, Joan Lewis-Wambi. IFITM1 overexpression enhances the aggressive phenotype of inflammatory breast cancer in a STAT2-dependent manner. [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 3560. doi:10.1158/1538-7445.AM2015-3560

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand crosslinks (ICL). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, interstrand crosslink repair protein, FANCA, greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner. Citation Format: Anaid Benitez, Fenghua Yuan, Satoshi Nakajima, Leizhen Wei, Liangyue Qian, Richard Myers, Jennifer J. Hu, Li Lan, Yanbin Zhang. FANCA regulates MUS81-EME1 mediated DNA incision in a damage-dependent manner. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 39. doi:10.1158/1538-7445.CANSUSC14-39

We previously reported that tumor suppressor LZAP is lost in a portion of head and neck squamous cell carcinomas and that LZAP inhibits NF-kB, while activating p53 in ARF-dependent and -independent fashions. The ability of LZAP to regulate p53 in the absence of ARF has been largely unexplored. Here, we show that LZAP depletion diminished expression of mutant and wild-type p53 protein. LZAP activity toward p53 was independent of ARF and the known p53 regulators, Wip1 and HDM2. Loss of LZAP decreased p53 translation and destabilized p53 protein due, at least partially, to increased nucleolin expression. Knockdown of LZAP abrogated p53 stabilization induced by ionizing radiation, and LZAP depletion protected wild-type p53 cells from radiation or chemotherapeutic agents, while rendering cells expressing mutant or no p53 more sensitive. In human HNSCC, LZAP levels correlated with p53 levels, and remarkably, tumors with low LZ were less likely to have p53 mutations. These data suggest that loss of LZAP represents a novel mechanism of p53 inactivation in human cancer. Given these findings, inhibition of LZAP activity toward p53 for patients with tumors harboring p53 mutations may represent a therapeutic strategy to simultaneously sensitize tumors while protecting normal tissues from radiation or chemotherapy. Citation Format: Dan Liu, Natalia Issaeva, Wendell G. Yarbrough. Loss of LZAP inactivates p53 in head and neck cancer and regulates sensitivity of cells to DNA damage in the p53-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1587. doi:10.1158/1538-7445.AM2013-1587

Leukemia stem cells (LSC) play a crucial role in initiation, maintenance, and progression of chronic myeloid leukemia (CML). Targeted therapy with a BCR-ABL targeted tyrosine kinase inhibitor (e.g. dasatinib) has improved survival of patients with chronic phase CML. However, like blast crisis (BC) CML, survival remains only 30% over 5 years, and treatment frequently fails to eliminate dormant LSC that are hypothesized to drive CML relapse. Overexpression of BCL2 family genes has been observed in human BC CML and may fuel LSC survival. Our previous data demonstrated that pro-survival long isoforms of BCL2 and MCL1 were preferentially expressed in BC CML LSC compared to normal stem cells. These data showed that the bone marrow (BM) is enriched for BCL2 and MCL1-expressing LSC and serves as a reservoir for therapeutic resistance, thereby underscoring the importance of pan-BCL2 inhibition in the BM. A potent pan-BCL2 inhibitor, sabutoclax, has been identified that inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl1-1 at nanomolar concentrations. To demonstrate whether sabutoclax or the combination of sabutoclax and dasatinib could synergize to eradicate LSC in the BM, humanized BC CML LSC mouse models were established by using 50,000 CD34+ cells selected from primary BC CML patient samples and transplanted into neonatal RAG2-/-γc-/- mice. Mice were screened for peripheral blood (PB) engraftment of human cells at 6 weeks, randomized, and dosed with (1) vehicle, (2) sabutoclax (10mg/kg, i.v., twice a week for 2 weeks), (3) dasatinib (50mg/kg, p.o., daily for 2 weeks), or (4) combination. Mice were sacrificed one day after last dose; PB, spleen (SP) and BM were collected for FACS analysis to measure engraftment and cell cycle status of LSC in different hematopoietic niches. BCL2 and MCL1 expression was measured in BM and SP by isoform specific qRT-PCR and immunohistochemistry. Treatment of CML LSC-transplanted mice with sabutoclax alone led to a significant reduction in LSC burden in the SP, but only slightly in the BM. Meanwhile, Single agent sabutoclax reduced the engraftment of quiescent LSC. The effect of sabutoclax synergized with dasatinib to further reduce LSC burden in the BM to eradicate LSC that drive therapeutic resistance. Importantly, these treatments spare normal human stem cells in both BM and SP thereby providing in vivo evidence of a reasonable therapeutic index. Sabutoclax not only reduced the expression of BCL2 and MCL1 mRNA long isoform in the BM, but also significantly reduced the number of human CD34+, BCL2+ and MCL1+ cells in both BM and SP. These results demonstrate that BM and SP niche BC CML LSC survival is driven by overexpression of multiple pro-survival BCL2 family isoforms rendering them susceptible to a novel pan-BCL2 inhibitor, sabutoclax, at doses that spare normal hematopoietic progenitors. Citation Format: Wenxue Ma, Janine Low-Marchelli, Heather S. Leu, Angela Court Recart, Jun Wei, Xianshu Huang, Maryla Krajewska, Maurizio Pellecchia, John C. Reed, Catriona H.M. Jamieson. BCR-ABL tyrosine kinase inhibition combined with a novel pan BCL2 family inhibitor abrogates leukemia stem cell survival in a niche-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2013-217

The mechanisms by which tumor cells metastasize and the role cell polarity proteins play in this process are not well understood. We report that Partitioning defective protein 3 (Par3) is dysregulated in metastasis in human breast cancer, and is associated with higher tumor grade and ErbB2-positive status. Downregulation of Par3 cooperated with ErbB2 to induce cell invasion and metastasis in vivo. Interestingly, the metastatic behavior was not associated with an overt mesenchymal phenotype. However, loss of Par3 inhibited E-cadherin junction maturation, disrupted membrane and actin dynamics at cell-cell junctions and decreased cell-cell cohesion in a Tiam1/Rac-GTP pathway dependent manner. Inhibition of this pathway restores E-cadherin junction maturation and blocked invasive behavior of cells lacking Par3 suggesting that loss of Par3 promotes metastatic behavior of ErbB2-induced tumor epithelial cells by decreasing cell-cell cohesion. Citation Format: Bin Xue, Senthil Muthuswamy. Loss of Par3 promotes metastasis of ErbB2 transformed mammary epithelial cells by inhibiting E-cadherin adhesion junction maturation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A58.

Defects in apoptosis contribute to poor outcome in high risk pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. “Inhibitor of Apoptosis” (IAP) proteins present promising targets, since they are expressed at high levels in acute leukemia. In the present study, we investigated the effect of small molecule IAP inhibitors alone and in combination with chemotherapeutics in ALL cell lines, primary leukemic blasts, normal peripheral blood lymphocytes and in a mouse model of pediatric ALL. Here, we report that IAP inhibitors at subtoxic concentrations cooperate with various anticancer drugs (i.e. AraC, Gemcitabine, Cyclophosphamide, Doxorubicin, Etoposide, Vincristine, Taxol) to induce apoptosis in ALL cells in a highly synergistic manner as calculated by combination index (CI index 0.3). Also, IAP inhibitors act in concert with AraC to reduce clonogenic survival, thus demonstrating also a long-term anti-leukemic effect. Importantly, we identify RIP1 as a critical regulator of this synergism of IAP inhibitors and AraC that mediates the formation of a RIP1/FADD/caspase-8 complex via an autocrine/paracrine loop of TNFα. Knockdown of RIP1 abolishes formation of this complex and subsequent activation of caspase-8 and -3, mitochondrial perturbations and apoptosis. Similarly, pharmacological inhibition of RIP1 kinase activity by Necrostatin-1 inhibits IAP inhibitor- and AraC-triggered interaction of RIP1, FADD and caspase-8 and apoptosis. In addition, blockage of TNFα by a TNFα-neutralizing antibody profoundly attenuates the cooperativity of IAP inhibitor and AraC to induce formation of the RIP1/FADD/caspase-8 complex and apoposis. Of note, IAP inhibitors potently trigger cell death in leukemic blasts from children with ALL ex vivo and also sensitize primary leukemia cells for chemotherapy-induced cell death. In contrast to malignant cells, IAP inhibitors and AraC at equimolar concentrations are non-toxic to both resting and activated normal peripheral blood lymphocytes, pointing to a therapeutic window. Most importantly, IAP inhibitors exert potent anti-leukemic activity in vivo in a mouse model of pediatric ALL engrafted in NOD/SCID mice. In conclusion, our findings provide first evidence that IAP inhibitors present a promising strategy to prime childhood ALL cells for chemotherapy-induced apoptosis in a RIP1-dependent manner. These data have important implications for the development of apoptosis-targeted therapies in childhood leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4692. doi:10.1158/1538-7445.AM2011-4692

Discussion Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-190.