Selected Articles from This Issue.

Objective: Ovarian cancer often presents with advanced stage disease. Despite surgery and chemotherapy, recurrence remains inevitable and prognosis poor. Advances in immunotherapy including programmed cell death-1 (PD-1) inhibition show promise, however clinical trials show modest response rates as a single agent therapy in ovarian cancer patients. Radiation therapy is an attractive strategy to enhance the efficacy of immune checkpoint blockade in ovarian cancer due to its ability to increase tumor-infiltrating lymphocytes and enhance immune activation. While ovarian cancer is known to be radiosensitive, total abdominal radiation is not routinely utilized due to high abdominopelvic toxicity. We have recently shown that ultra-high dose rate FLASH irradiation reduces intestinal radiation-induced toxicity. Importantly, we demonstrated efficient tumor control in preclinical models of ovarian cancer peritoneal metastasis. Here we sought to determine the efficacy and safety of FLASH irradiation combined with PD-1 inhibition in preclinical models of ovarian cancer. Methods: Female C57BL/6 mice were intraperitoneally inoculated with ID8-Asc or UPK10 ovarian cancer cells. Total abdominal FLASH irradiation at 14 Gy was administered 10 days post-inoculation. Anti-PD-1 and isotype control antibodies were injected on days 7, 10, and 13 post-inoculation. Mice were sacrificed 22-27 days post-inoculation for tumor burden and immune cell analysis. Stool count, stool weight, complete blood count, and survival were evaluated to compare acute and chronic toxicity as well as efficacy in the treatment arms. Results: Four cohorts of mice were analyzed: Isotype control antibody (IgG), anti-PD-1 (aPD-1), FLASH + IgG, and FLASH + aPD-1. In the ID8-Asc model, irradiated mice showed a 44% decrease in solid stool production with subsequent recovery to baseline by 14 days post-treatment. There was no evidence of hematopoietic toxicity in any of the treatment arms. Survival analysis is ongoing. Exploratory necropsy demonstrated significant tumor burden control in the FLASH cohorts, and in particular, a 25-fold decrease in tumor number in the FLASH + aPD-1 cohort compared to control (mean tumor weight 1.82 mg vs. 357.2 mg, respectively). FLASH + aPD-1 increased intratumoral CD4+ and CD8+ T cells and correlated with enhanced tumor control compared to FLASH or aPD-1 single agent treatments. Similar results were observed in the UPK10 model. Conclusion: Combination FLASH and PD-1 inhibition enhanced tumor control and recruitment of tumor infiltrating lymphocytes compared to IgG, aPD-1, and FLASH treatments alone, while demonstrating safety and tolerability. Our data identify a new opportunity for FLASH and checkpoint inhibition in the treatment of ovarian cancer. Citation Format: Stephanie Chow, Joshua T. Eggold, Karen Levy, Jinghui Wang, Rakesh Manjappa, Dylan Y. Breitkreutz, Amy S. Yu, Karl Bush, Oliver Dorigo, Billy W. Loo, Erinn B. Rankin. Total abdominal ultra-rapid FLASH irradiation enhances the efficacy of PD-1 inhibition in preclinical models of ovarian cancer [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 5357.

Introduction: We report on the development and preclinical validation of NBE-002, a next-generation antibody drug conjugate (ADC) for the treatment of triple-negative breast cancer (TNBC). NBE-002 consists of a humanized monoclonal antibody directed against the receptor tyrosine kinase ROR1, expressed on the surface of numerous solid cancers including TNBC, and is site-specifically conjugated to a derivative of the highly potent anthracycline PNU-159682. Results: Therapeutic efficacy of NBE-002 was evaluated in ROR1-low/-intermediate/-high patient-derived xenograft (PDX) models of TNBC, lung adenocarcinoma, ovarian carcinoma, and a variety of sarcomas. NBE-002 was found to display significant anti-tumor activity in each indication. The most pronounced effect was achieved in TNBC, where complete tumor regression was observed already at the lowest ADC dose tested (0.33 mg/kg), even in models expressing low levels of ROR1 (H-score ≤70). Since these PDX studies were performed in immune-compromised hosts, and anthracyclines are known inducers of immunogenic cell death, anti-tumor activity was also investigated in syngeneic tumor models in immune-competent hosts. Anti-tumor activity of PNU-ADCs involved activation of the immune system, as shown by evaluation of NBE-002 or a Trastuzumab-PNU conjugate (T-PNU) in ROR1- or HER2-positive syngeneic breast cancer models, respectively. Depletion of CD8 T cells severely reduced anti-tumor activity, demonstrating an important role for T cells in driving tumor regression. Furthermore, when tumor free animals were re-challenged with the same tumor, tumor growth was inhibited in the absence of any further ADC administration, indicating the development of an immunological memory. Notably, combination of ADC and checkpoint inhibition, such as α-PD1 or α-CTLA4, significantly enhanced tumor eradication following the treatment. Conclusion: Our results demonstrate that NBE-002 is a highly effective and promising targeted therapeutic for the treatment of ROR1 positive TNBC and potentially other solid tumor indications that warrants clinical development. Considering the pronounced immune-modulatory functions of the PNU payload, NBE-002 may be particularly well suited for combination therapy with immune checkpoint inhibitors. NBE-002 is currently undergoing GMP manufacturing and initiation of clinical studies is expected in mid-2020. Citation Format: Roger R. Beerli, Lorenz Waldmeier, Rémy Gébleux, Francesca Pretto, Ulf Grawunder. NBE-002, an anthracycline-based immune-stimulatory antibody drug conjugate (iADC) targeting ROR1 for the treatment 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 LB-197.

Background: Triple-negative breast cancer (TNBC) requires new treatment options due to its aggressive nature, high recurrence rates with standard-of-care treatment, and heterogeneous therapeutic effectiveness of existing treatments for metastatic TNBC. Poly(ADP-ribose) polymerase (PARP) inhibitors are approved treatments for HER2-negative breast cancer with germline BRCA (gBRCA) mutation, and they are of particular interest for patients with gBRCA-mutated TNBC due to their role in exploiting DNA repair deficiencies. Two PARP inhibitors (olaparib and talazoparib) are FDA-approved for patients with metastatic gBRCA mutated breast cancer. Although PARP inhibitor treatment has high response rate and improved progression-free survival, resistance to PARP inhibitors poses an emerging and significant challenge, necessitating the identification of novel therapeutic targets to overcome this resistance and improve patient outcomes with gBRCA mutated TNBC. This study aims to identify novel therapeutic targets whose inhibition could enhance the efficacy of PARP inhibitors in TNBC and olaparib-refractory TNBC. Methods: We conducted a preclinical experiment to achieve our aim. We generated olaparib-resistant (OPR) cell lines using BRCA-mutated TNBC cell lines (SUM149, HCC1937, and MDA-MB-436). These cell lines were treated with olaparib until they achieved at least a 10-fold higher concentration of IC50 than their parent cell lines. To identify potential synergistic kinase targets, we performed a non-biased high-throughput 709 Kinome-Library RNA interference assay using SUM149-OPR cells. Target candidates were selected using the Sensitivity Index (SI > 0.12) and STRING interactome analysis. We then conducted a series of assays, including a sulforhodamine B viability assay, a soft-agar assay, and a Bliss Synergy Score analysis, to determine the combination effect of selected kinase inhibitors and olaparib using SUM149-OPR, HCC1937-OPR, and MDA-MB-436-OPR cell lines. Finally, we conducted a soft-agar assay to evaluate in vitro tumorigenicity. Results: Kinome-Library RNA interference screening revealed that the DNA damage pathway (ATR, CDK12, etc.) and phosphoinositide 3-kinases pathway (PIK3CG, PIK3R3, PIK3R4, etc.) as potential canonical pathways targets for synergizing with olaparib efficacy. PLK4 was selected as non-canonical target. Based on target identification, we selected inhibitors of ATR (elimusertib, gartisertib), ATM (AZD1390), PLK4 (CFI-400945), CDK12 (SR-4835), PI3KCA (copanlisib), and AKT (capivasertib) for proliferation assays. We found that targeting the DNA damage repair pathway was the most effective in enhancing the efficacy of olaparib in both parent and olaparib-resistant TNBC cell lines. Bliss score ranges are 5.509 to 48.763 for elimusertib (ATR inhibitor), 7.872 to 71.406 for gartisertib (ATR inhibitor), and 4.660 to 45.836 for AZD1390 (ATM inhibitor). Further, we observed a significantly enhanced in vitro antitumor effect of the olaparib and elimusertib combination (P<0.05). Conclusions: Our preclinical study identified DNA repair pathways as potential targets to enhance the efficacy of olaparib in both parent and olaparib-refractory gBRCA mutated TNBC cell lines. Future in vivo studies, including patient-derived xenografts and additional mechanistic analyses to further understand dual DNA targeting synergy, are warranted to validate this therapeutic approach for TNBC with gBRCA mutation. Citation Format: Diana Hawkins-Westergard, Anya Dimitrijevic, Clinton Yam, Rachel M. Layman, Naoto T. Ueno, Banu Arun, Jangsoon Lee. Overcoming Olaparib Resistance in BRCA-Mutated Triple-Negative Breast Cancer: Synergistic Potential of DNA Repair Pathway Inhibitors [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-08-22.

Simple SummaryTriple negative breast cancer (TNBC) represents 15 to 20% of all breast cancers in the United States. The main treatment option remains chemotherapy, despite limited efficacy. New biologic and targeted agents are increasingly emerging for the treatment of TNBC. Given the continuous advances in the field of TNBC, this review assesses the latest developments in basic characterization, subtyping, and treatment of TNBC, including novel drug developments with antibody-drug conjugates, immune checkpoint inhibitors, PARP inhibitors, and androgen receptor targeted agents.Breast cancer (BC) is the most common malignancy affecting women. It is a highly heterogeneous disease broadly defined by the differential expression of cell surface receptors. In the United States, triple negative breast cancer (TNBC) represents 15 to 20% of all BC. When compared with other subtypes of BC, TNBC tends to present in younger women, and has a higher mortality rate of 40% in advanced stages within the first 5 years after diagnosis. TNBC has historically had limited treatment options when compared to other types of BC. The mainstay of treatment for TNBC remains cytotoxic chemotherapy despite the emergence of new biologic and targeted agents. Defining the specific tumor molecular profile including PDL-1 and androgen receptor testing is expanding treatment options in the clinical setting. Identifying more targetable, novel biomarkers that may better define therapeutic targets or prognostic markers is currently underway. TNBC nomenclature is expected to be updated in favor of other nomenclature which would help direct therapy, and further redefine TNBC’s heterogeneity. Given the continuous advances in the field of TNBC, this review assesses the latest developments in basic characterization, subtyping, and treatment of TNBC, including novel drug developments with antibody-drug conjugates, immune checkpoint inhibitors, PARP inhibitors and androgen receptor targeted agents. Future trials are necessary in the face of these innovations to further support the use of new therapies in TNBC and the detection of the appropriate biomarkers.

Introduction: Selinexor (KPT-330) is a first-in-class oral exportin-1 (XPO1/ CRM1) inhibitor that induces cell cycle arrest and apoptosis in cancer cells through reactivation of tumor suppressor proteins and inhibition of DNA damage repair genes. Here, we studied selinexor in combination with niraparib, an inhibitor of the DNA damage repair proteins PARP1 and 2, in preclinical models of ovarian cancer. Given that both compounds can inhibit DNA damage repair responses, we hypothesized the combination of selinexor and niraparib would enhance cancer cell death by accumulation of DNA damage that cannot be resolved in ovarian cancer. Methods: Selinexor and niraparib alone and in combination were tested in vitro on the BRCA wildtype ovarian cancer cell line A2780. Total RNA and protein were extracted from cell lysates and analyzed by qPCR and immunoblots. In vivo, a subcutaneous A2780 xenograft mouse model was treated with selinexor [10 mg/kg, once per week (M) for three weeks] or niraparib [37.5 mg/kg, once-daily for five days per week (M-F) for three weeks] as single agents or in combination. Tumor growth and body weights were measured for 21 days. Tumors were harvested at the end of the study and analyzed by immunohistochemistry (IHC). Results: Selinexor and niraparib as single agents inhibited A2780 cell proliferation (selinexor IC50: 200nM; niraparib IC50: 800nM). The combination of selinexor and niraparib showed synergistic cytotoxicity in A2780 cells. Increased expression of phospho (S139) H2A.X with the combination confirmed induction of DNA damage. In vivo, the combination enhanced tumor inhibition (51.0% in combination versus 24.3% and 29.6% in selinexor and niraparib respectively) and improved median survival compared to vehicle or each agent alone (18 days in combination group versus 11, 14 and 16 days in vehicle, selinexor, and niraparib groups, respectively). IHC analysis showed enhanced nuclear p53 and p21 staining in selinexor treated tumors as well as in tumors treated with the combination. Increased apoptosis was observed in tumor samples treated with both selinexor and niraparib as compared to vehicle control or each agent alone. Conclusions: Selinexor plus niraparib demonstrated enhanced anti-tumor activity in preclinical models of human ovarian cancer. Since both drugs could inhibit DNA damage repair pathway, the drug combination efficiently accumulated DNA damage, which was associated with reduced cell proliferation, and induced apoptosis. This combination therapy warrants further investigation as a treatment option for patients with ovarian cancer. Citation Format: Hua Chang, Trinayan Kashyap, Sophie Debler, Thaddeus J. Unger, Sarah Wang, Keith Mikule, Jing Wang, Mansoor R. Mirza, Sharon Shacham, Yosef Landesman. Enhanced anti-tumor effects of selinexor and niraparib in preclinical models of ovarian cancer [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 5826.

Until very recently, the treatment for locally advanced or metastatic breast cancer revolved solely around sequential chemotherapies, alone or in combination. With the increasing recognition that triple negative breast cancers are a very heterogeneous group of tumors, exciting new strategies have emerged. Here we review some of the recent advances to standard chemotherapy now in clinical use as well discuss new strategies in development. Immunotherapy: There is currently an indication for nab-paclitaxel and atezolizumab for patients with locally advanced or metastatic triple negative breast cancer with PD-L1 positive tumor-infiltrating immune cells. The Impassion130 study first presented a median PFS of 7.2 months with nab-paclitaxel and atezolizumab vs. 5.5 months of nab-paclitaxel and placebo (HR 0.80; 95% confidence interval (CI) 0.69-0.92). Further analyses showed no benefit in the PD-L1 negative group.1 An exploratory overall survival analysis in the PD-L1+ subset showed an increase in the atezolizumab group of 25 months vs 18 in the control group.2 Immunotherapy for metastatic breast cancer will be presented in greater detail in another talk in this session. Sacituzumab govitecan: Sacituzumab govitecan is an antibody-drug conjugate (ADC) targeting Trop-2 and linked with SN-38, the active metabolite of irinotecan and a potent DNA- damaging agent. Trop-2 has been estimated to be expressed in up to 90% of TNBC, making it an attractive target for an ADC. Sacituzumab govitecan was conditionally approved for use after the Phase I/II trial demonstrated and objective response rate of 33.3% and a median overall survival of 13.0 months.3 The recently presented phase III ASCENT trial evaluated patients with metastatic TNBC who had at least 2 previous lines of therapy in the metastatic setting. There was an improvement in median PFS from 1.7 to 5.6 months for patients who received sacituzumab vs. physician’s choice of therapy. The HR for PFS was 0.41; 95% CI: 0.32-0.52) Additionally, there was an improvement in median OS of 6.7 to 12.1 months (HR 0.48; 95%CI:0.38-0.59).4PARP Inhibitors: There are now 2 PARP inhibitors that have FDA approval for use for patients with metastatic breast cancer and a germline BRCA pathogenic variant.5,6 Both Olaparib and Talazoparib demonstrated improvements in PFS which led to FDA approval. While neither the OlympiAD (olaparib) nor the EMBRACA (talazoparib) trial showed statistically significant improvement in OS, both trials showed a significant improvement in quality of life measures. Future Directions: Ongoing trials evaluating further ADCs, targeted therapies and drugs targeting DNA Damage Repair pathways for both germline and somatic pathogenic variants will be discussed 1.Schmid P, Adams S, Rugo HS, et al: Atezolizumab and Nab-Paclitaxel in Advanced Triple-Negative Breast Cancer. NEJM 379:2108-2121, 20182.Schmid P, Rugo HS, Adams S, et al: Atezolizumab plus nab-paclitaxel as first-line treatment for unresectable, locally advanced or metastatic triple-negative breast cancer The Lancet Oncology 21:44-59, 20203.Bardia A, Mayer IA, Diamond JR, et al: Efficacy and Safety of Anti-Trop-2 Antibody Drug Conjugate Sacituzumab Govitecan (IMMU-132) in Heavily Pretreated Patients With Metastatic Triple-Negative Breast Cancer. J Clin Oncol 35:2141-2148, 20174.Bardia A, Tolaney S, Loirat D, et al: ASCENT: A randomized phase III study of sacituzumab govitecan (SG) vs treatment of physician’s choice (TPC) in patients (pts) with previously treated metastatic triple-negative breast cancer (mTNBC). Annals of Oncology 31:S1142-S1215. 10.1016/annonc/annonc325, 20205.Robson M, Im S-A, Senkus E, et al: Olaparib for Metastatic Breast Cancer in Patients with a Germline BRCA Mutation. NEJM 377:523-533, 20176.Litton JK, Rugo HS, Ettl J, et al: Talazoparib in Patients with Advanced Breast Cancer and a Germline BRCA Mutation. NEJM 379:753-763, 2018 Citation Format: J Litton. Metastatic TNBC - What’s new on the horizon? [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP085.

Abstract. Statistics show that, second only to lung cancer, breast cancer is now the most common type of cancer worldwide. Compared to other forms of breast cancer, triple negative breast cancer (TNBC) has a worse prognosis, extensive metastases, and poor efficacy, making treatment more difficult. antibody-drug conjugates (ADCs), have become a hot topic in targeted tumor therapy research. Gosa tuzumab is the first drug approved for advanced TNBC treatment globally. However, challenges remain in the clinical use of ADCs, such as limited delivery of ADCs to target cells and suboptimal anti-tumor effects. This article analyzes targeted therapy methods and ADCs for TNBC, summarizes current market gosa tuzumab and ongoing clinical trials for ADCs drugs, providing a reference for further study on TNBC treatment with ADCs. Nevertheless, issues like drug resistance, adverse reactions and combination strategies with other chemotherapy inhibitors for TNBC treatment warrant further investigation in future clinical trials.

<div>Abstract<p><b>Purpose:</b> Triple-negative breast cancer (TNBC) is a highly heterogeneous disease and has the worst outcome among all subtypes of breast cancers. Although PARP inhibitors represent a promising treatment in TNBC with <i>BRCA1</i>/<i>BRCA2</i> mutations, there is great interest in identifying drug combinations that can extend the use of PARP inhibitors to a majority of TNBC patients with wild-type <i>BRCA1</i>/<i>BRCA2</i>. Here we explored whether mTOR inhibitors, through modulating homologous recombination (HR) repair, would provide therapeutic benefit in combination with PARP inhibitors in preclinical models of BRCA-proficient TNBC.</p><p><b>Experimental Design:</b> We have studied the effects of mTOR inhibitors on HR repair following DNA double-strand breaks (DSB). We further demonstrated the <i>in vitro</i> and <i>in vivo</i> activities of combined treatment of mTOR inhibitors with PARP inhibitors in BRCA-proficient TNBC. Moreover, microarray analysis and rescue experiments were used to investigate the molecular mechanisms of action.</p><p><b>Results:</b> We found that mTOR inhibitors significantly suppressed HR repair in two BRCA-proficient TNBC cell lines. mTOR inhibitors and PARP inhibitors in combination exhibited strong synergism against these TNBC cell lines. In TNBC xenografts, we observed enhanced efficacy of everolimus in combination with talazoparib (BMN673) compared with either drug alone. We further identified through microarray analysis and by rescue assays that mTOR inhibitors suppressed HR repair and synergized with PARP inhibitors through regulating the expression of SUV39H1 in BRCA-proficient TNBCs.</p><p><b>Conclusions:</b> Collectively, these findings strongly suggest that combining mTOR inhibitors and PARP inhibitors would be an effective therapeutic approach to treat BRCA-proficient TNBC patients. <i>Clin Cancer Res; 22(7); 1699–712. ©2015 AACR</i>.</p></div>

Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of invasive breast cancer that lacks ER, PR, and HER2 expression. It is a heterogeneous disease with several molecular subtypes: basal-like1 (BL-1), basal-like 2 (BL-2), mesenchymal (M), and luminal androgen receptor (LAR). Treatment for TNBC is normally limited to chemotherapy, and relapse is common. Here we report molecular alterations associated with TNBC metastasis by analyzing the genomic profiles of paired primary and metastatic TNBCs. Methods:50 paired TNBCs were identified through an IRB-approved protocol via the City of Hope (COH) Biospecimen Repository. DNA mutation and mRNA expression profiles of 10 paired primary and metastatic TNBCs were analyzed. DNA mutations were identified using exome sequencing by FoundationOne®. Affymetrix Human Genechip 2.0 was used for mRNA expression profiling. Raw data were normalized and processed using Expression Console, and linear regression was performed using Limma to identify the differentially expressed genes between primary and metastatic TNBCs. Results: DNA mutation profiling showed that multiple mutations occurred within genes covering pathways of PI3K/AKT/mTOR, DNA repair, RAS/MAPK, cell cycle, and growth factor receptor signaling, reconfirming genomic heterogeneity of TNBCs. Gene expression profiles were analyzed for Lehmann's TNBC molecular subtypes (BL-1, BL-2, M, and LAR). Six of ten TNBCs showed phenotype shift between the primary and metastatic TNBCs. Several unique gene expression patterns were identified by comparing the paired TNBCs. CCNE1 and TPX2 were co-overexpressed in metastatic TNBCs compared to paired primaries. This mirrored prior studies in ovarian cancer, where co-overexpression of CCNE1 and TPX2 were found related to clonal resistance against chemotherapy. Splicing factors TRA2B and SRSF7 were also over-expressed in metastatic TNBCs compared with primaries. The analysis studying the association of CCNE1 and TPX2 with overall survival is ongoing using TCGA. Conclusion: Overall, these results show the comparative changes between primary and relapsed TNBCs and indicate the heterogeneity of molecular mechanisms of recurrence. CNNE1 and TPX2 may represent important genes involved in TNBC metastasis. Further analyses including a total of 50 paired TNBCs are currently underway. Study Contact: Yuan Yuan MD PhD, City of Hope Comprehensive Cancer Center; Duarte, CA 91030; Email: yuyuan@coh.org Citation Format: Zhao Z-M, Yost S, Yuan Y-C, Warden C, Chuang J, Yuan Y. Mechanisms of recurrence: Paired analysis of primary and metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-02.

Introduction Novel compounds have entered the triple-negative breast cancer (TNBC) treatment algorithm, namely immune checkpoints inhibitors (ICIs), PARP inhibitors and antibody-drug conjugates (ADCs). The optimization of treatment efficacy can be enhanced with the use of combination treatments, and the incorporation of novel compounds. In this review, we discuss the combination treatments under development for the treatment of TNBC. Areas covered The development of new drugs occurring in recent years has boosted the research for novel combinations to target TNBC heterogeneity and improve outcomes. ICIs, ADCs, tyrosine kinase inhibitors (TKIs), and PARP inhibitors have emerged as leading players in this new landscape, while other compounds like novel intracellular pathways inhibitors or cancer vaccines are drawing more and more interest. The future of TNBC is outlined in combination approaches, and based on new cancer targets, including many chemotherapy-free treatments. Expert opinion A large number of TNBC therapies have either proved clinically ineffective or weighted by unacceptable safety profiles. Others, however, have provided promising results and are currently in late-stage clinical trials, while a few have actually changed clinical practice in recent years. As novel, more and more selective drugs come up, combination strategies focusing the concept of synergy are fully warranted for the future.

Poly (ADP-ribose) polymerase (PARP) inhibitors represent one of the most exciting recent developments in cancer therapy. While substantial efficacy has been shown with clinically available PARP inhibitors (PARPis), to date, in treatment of hereditary deletions of BRCA1/2 in breast and ovarian cancers, the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTis), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPis) as a treatment strategy for acute myelogenous leukemias (AML) and triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPis to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMTs led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPis (e.g. BMN 673) appears to correlate with the degree of trapping of PARP1 in chromatin. We first find that in cultured human AML and TNBC cells, the DNMTis (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPis (1 to 10 nM BMN 673) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment of TNBC cell line MDA-MB-231 resulted in significantly enhanced retention of PARP1 and DNMT1 at sites of double strand breaks (DSBs) induced by laser microirradiation. Concomitant with this, the combined doses resulted in significant increases in cytotoxic DSBs, observed 4-24 hours after DSB induction, when compared to single-drug treatments. Homologous recombination (HR) DSB repair activity also appears decreased, as measured by GFP reporter assays. In keeping with these findings, colony survival assays demonstrated that the combination treatment, compared to either drug alone, strongly inhibited colony formation of TNBC cell lines (N=4). Notably non-tumorigenic MCF10A cells showed no significant differences in colony numbers with single or combination drug treatments. Similar to TNBCs, AML cell lines (N=3) as well as primary AML cells (N=8) showed dramatic decreases in colonies in combination vs single agent drug treatments. In the most important translational implications of the preliminary studies, in in vivo therapy TNBC and AML models in immune-deficient mice, our low dose combinations of DNMTis and PARPis provide for potent anti-tumor responses. Mouse xenograft experiments using BRCA mutant TNBC cell line SUM149PT demonstrated that the combination treatment has a significant (p<0.05) survival advantage compared to control (vehicle), AZA (0.5mg/kg) or BMN (0.3 mg/kg) alone. Importantly, mouse MDA-MB-231 xenografts with intact BRCA1 showed significant survival with the combined drug treatment. Likewise in AML xenografts of MOLM14 and MV411 cell lines treated with the drug combination show significantly decrease leukemia burden, as measured by luciferase imaging. Our data suggest a novel use of both DNMTis and PARPis in a compelling therapeutic strategy for TNBCs independent of BRCA mutations and poor prognosis AML; the latter will be investigated in a clinical trial to be based at the University of Maryland and funded by Van Andel-SU2C. Citation Format: Nidal Muvarak, Khadiza Chowdhury, Carine Robert, Xia Limin, Eun Yong Choi, Yi Cai, Marina Bellani, Michael Seidman, Maria R. Baer, Rena Lapidus, Stephen B. Baylin, Feyruz V. Rassool. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for multiple cancers: Key data in AML and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA13.

monotherapy in advanced cancer patients with a germline BRCA1/2 mutation. Among other cancers, the study includes an ovarian cancer cohort of 193 patients with platinum-resistant or platinum-unsuitable ovarian cancer and a cohort of 62 patients with breast cancer. Patients with ovarian cancer were eligible if they were platinum resistant, or if they were not suitable for further platinum therapy due to hypersensitivity or toxicity as deemed by their primary oncologist. Percentage of patients fitting this unsuitability criterion has not been prospectively collected and reported. Data regarding if or what percentage of ovarian cancer patients had a high-grade serous histology have not been provided. Also, data on the percentage of patients with breast cancer with triple-negative disease is not available. The primary end point of the study was tumor response rate by RECIST criteria for all patients. CA-125 levels have not been provided to correlate for response in ovarian cancer cohort. Among patients with platinum-sensitive relapsed ovarian cancer, olaparib therapy has been shown to improve progression-free survival regardless of the BRCA mutational status. 2 The efficacy of olaparib may depend on platinum sensitivity even among patients with BRCAmutated ovarian cancer suggesting that platinum sensitivity may be more relevant to predict response to poly(ADP–ribose) polymerase (PARP) inhibitors than the mutational status of BRCA1 and BRCA2. 3 Many genes other than BRCA are involved in platinum-induced DNA damage repair and are related to platinum sensitivity. 4-5 Not knowing exactly what percentage of patients in Kaufman et al study were platinum resistant versus platinum unsuitable makes it less clear if more than 30% response rate for ovarian cohort were due to BRCA mutation or platinum sensitivity. In other tumor types response rates according to prior platinum therapy also favored no prior platinum exposure even though it did not reach statistical significance. However, the sample sizes were small and, as stated by Kaufman et al, 1 the CIs overlapped. In the Cancer Genome Atlas project 6 approximately 22% of tumors had a BRCA mutation, which is associated with homologous recombination deficiency (HRD), the hallmark for response to PARP inhibitors. The high grade serous ovarian cancers and triple negative breast cancers may represent histologically and phenotypically defined subgroups associated with HRD. The data regarding the percentage of these patients in these two subgroups in ovarian and breast cancer cohorts would be helpful if available. It remains to be shown what exactly determines the response to PARP inhibitor therapy; Is it germline BRCA1/2 mutations? Is it platinum sensitivity? Is it HRD? Or is it a combination of all? Currently, identification of germline BRCA mutations through a blood test seems to be the only US Food and Drug Administration–approved surrogate marker for HRD. However testing tumor tissue for somatic BRCA mutations as well as for genes associated with HRD may better define the subgroup of patients with cancer who would benefit from a number of PARP inhibitors yet to come into clinical practice. The recent US Food and Drug Administration approval of olaparib for ovarian cancer along with a companion diagnostic blood test of BRACAnalysis CDx (Myriad Genetics Laboratories, Salt Lake City, UT) seems to be just the beginning.

Challenging Clinical Scenarios: Treatment of Patients With Triple-Negative or Basal-Like Metastatic Breast Cancer

Trophoblast cell surface antigen 2 (Trop-2) is overexpressed on many epithelial carcinomas, yet is expressed at much lower level on normal tissue. Overexpression of Trop-2 has been correlated with poor prognosis in several solid tumors. These two characteristics make Trop-2 a potential drug target. An antibody-drug conjugate (ADC) targeting Trop-2, IMMU-132, has recently been demonstrated to be effective in treating triple negative breast cancer (TNBC) and gastric cancer patients. Here we present a Trop-2 ADC, BAT8003, which contains an uncleavable linker and a maytansine derivative as the payload. An A114C mutation on antibody heavy chain was introduced to BAT8003 for site-specific conjugation, in order to generate a more homogeneous product for a better pharmacokinetics profile. BAT8003 is also completely devoid of fucose modification, to allow for enhanced ADCC effect. We show that BAT8003 is effectively internalized upon binding to Trop-2, and inhibits proliferation of Trop2-overexpressed tumor cells with IC50s of ˜1 nM. In a TNBC (MDA-MB-468 cell) mouse xenograft model, BAT8003 strongly inhibits tumor growth at a dose level as low as 5 mg/kg. BAT8003 also demonstrates potent activity in another TNBC (MX-1 cell) mouse tumor model, in which it shows the same inhibition activity as the naked antibody conjugated heterogeneously with almost two fold payload (DAR 3.5), suggesting the effect caused by site-specific conjugation. We are currently developing BAT8003 for clinical evaluation in TNBC and other cancer indications. Citation Format: Tang W, Huang X, Ou Z, Yan H, Gan J, Dong Q, Tan B, Yang Y, Guo Y, Li S, Thomas B, Yu J-C. BAT8003, a potent anti-Trop-2 antibody-drug conjugate, for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-16.

Background and Aims: Triple-negative breast cancer (TNBC) is the most aggressive subtype, and is often associated with mutations in the breast cancer susceptibility protein (BRCA) genes, which are known tumor-suppressor genes involved in DNA damage repair. Chromosomal instability (CIN) has been added to the hallmarks of cancer and is characterized by a frequent gain or loss of chromosomes during mitosis (i.e. chromosomal aberrations). BRCA1 and BRCA2 breast tumors develop by specific and distinct evolutionary paths, as their gene profiles and genome aberration spectra differ from each other and from those in sporadic BC. The lncRNA Non-Coding RNA Activated by DNA Damage (NORAD) has been demonstrated to contribute to the progression of several cancer entities by regulating genomic stability. Therefore, we wanted to explore a potential additive anti-cancerous effect by treating TNBC cells with a combination of NORAD silencing and PARP inhibition. Material and Methods: To clarify the potential clinical significance of NORAD in human TNBC, patient data from publicly available databases were analyzed. In order to further characterize NORAD, we performed phenotypic experiments after establishing an siRNA-mediated knock-down approach. Expression levels of NORAD as well as DNA-damage repair proteins were analyzed by qRT-PCR and Western Blot after combined cell treatment with NORAD silencing and PARP inhibition. To further unravel the molecular mechanism behind, we performed immunofluorescent experiments against markers for DNA-double strand breaks and homologous recombination efficiency (i.e. Rad51 and yH2AX) after TNBC treatment with NORAD siRNAs and PARP inhibitors. Results: High NORAD levels were identified to be a negative prognostic factor for disease-free survival of TNBC patients (HR with 95% CI: 2.4 (1.35-4.25); n=161; p=0.002). Furthermore, the NORAD gene is amplified in up to 3% of BC cases and mRNA levels are higher in tumor tissue when compared to healthy tissue. A qPCR based screening of several breast cancer cell lines showed an at least 4-times increased expression of NORAD when compared to normal mammary cells (i.e. MCF 12A). Knock-down of NORAD in TNBC cells resulted in a slightly reduced proliferation as well as to a decreased expression of several DNA repair proteins. Immunofluorescent experiments of TNBC cells treated with the PARP inhibitor olaparib and/or after NORAD silencing revealed increased yH2AX-dependent nuclear foci formation when compared to control conditions, indicating augmented DNA double-strand breaks after treatment. Conclusion: Our first data let us hypothesize that a combination of NORAD knock-down and PARPi treatment increases inhibition of DNA repair mechanisms and leads to a more pronounced “anti-tumorigenic” phenotype in TNBC cells. This approach could lead to the optimization of current treatment concepts. Furthermore, as increased NORAD expression is associated with an unfavorable patient survival, this lncRNA could serve as potential prognostic biomarker. Citation Format: Christiane Klec, Anita Kapeller, Felix Prinz, Christoph Trenk, Herbert Stöger, Martin Pichler. Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer [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 P1-05-13.