Jamunarani Veeraraghavan

Recurrent ESR1 – CCDC170 rearrangements in an aggressive subset of oestrogen receptor-positive breast cancers

Characterizing the genetic alterations leading to the more aggressive forms of estrogen receptor positive (ER+) breast cancers are of critical significance in breast cancer management. Here we identify recurrent rearrangements between estrogen receptor gene ESR1 and its neighbor CCDC170, which are enriched in the more aggressive and endocrine-resistant luminal-B tumors, through large-scale analyses of breast cancer transcriptome and copy number alterations. Further screening of 200 ER+ breast cancers identifies eight ESR1-CCDC170 positive tumors. These fusions encode N-terminally truncated CCDC170 proteins (ΔCCDC170). When introduced into ER+ breast cancer cells, ΔCCDC170 leads to markedly increased cell motility and anchorage-independent growth, reduced endocrine sensitivity, and enhanced xenograft tumor formation. Mechanistic studies suggest that ΔCCDC170 engages Gab1 signalosome to potentiate growth factor signaling and enhance cell motility. Together, this study identifies neoplastic ESR1-CCDC170 fusions in a more aggressive subset of ER+ breast cancer, which suggests a new concept of ER pathobiology in breast cancer.

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies. Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition. Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo. Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

De-escalation of treatment in HER2-positive breast cancer: Determinants of response and mechanisms of resistance

Overexpression and/or gene amplification of HER2, a crucial member of the HER family of four receptors, occur in about 15-20% of breast cancers and define an aggressive subtype of the disease. Activated HER homo and heterodimers govern a complex and redundant downstream signaling network that regulates cell survival and metastasis. Despite treatment with effective HER2-targeted therapies, many HER2-positive tumors fail to respond, or initially respond but eventually develop resistance. One of the upfront reasons for this treatment failure is failure to accurately select the tumors that are truly dependent on HER2 for survival and so would benefit the most from HER2-targeted therapy. In these truly HER2-addicted tumors (i.e. physiologically dependent), resistance could be the result of an incomplete inhibition of signaling at the HER receptor layer. In this regard, preclinical and clinical studies have documented the superiority of combination anti-HER2 therapy over single agent therapy to achieve a more comprehensive inhibition of the various HER receptor dimers. HER2 can be further activated or reactivated by mutations or other alterations in HER2 itself, or in other HER family members. Even when a complete and sustained HER inhibition is achieved, resistance to anti-HER therapy can arise by other somewhat dominant mechanisms, including preexisting or emerging alternative signaling pathways such as the estrogen receptor, deregulated downstream signaling components, especially of the PI3K pathway, and the tumor immune microenvironment. Most of the clinical trials that have investigated the efficacy of anti-HER2 therapies took place in the background of aggressive chemotherapy regimens, thus confounding the identification of key factors of resistance to the anti-HER2 treatments. Recent studies, however, have suggested that some HER2-amplified tumors may benefit from anti-HER2 therapy combined with only a single chemotherapy agent or in the absence of any chemotherapy. This de-escalation approach, a promising therapeutic strategy, is currently being explored in the clinic. In this review, we summarize the major molecular determinants that play a crucial role in influencing tumor response and resistance to HER2-targeted therapy, and discuss the growing need for patient stratification in order to facilitate the development of de-escalation strategies using HER2-targeted therapy alone with no chemotherapy.

Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

More aggressive and therapy-resistant oestrogen receptor (ER)-positive breast cancers remain a great clinical challenge. Here our integrative genomic analysis identifies tousled-like kinase 2 (TLK2) as a candidate kinase target frequently amplified in ∼10.5% of ER-positive breast tumours. The resulting overexpression of TLK2 is more significant in aggressive and advanced tumours, and correlates with worse clinical outcome regardless of endocrine therapy. Ectopic expression of TLK2 leads to enhanced aggressiveness in breast cancer cells, which may involve the EGFR/SRC/FAK signalling. Conversely, TLK2 inhibition selectively inhibits the growth of TLK2-high breast cancer cells, downregulates ERα, BCL2 and SKP2, impairs G1/S cell cycle progression, induces apoptosis and significantly improves progression-free survival in vivo. We identify two potential TLK2 inhibitors that could serve as backbones for future drug development. Together, amplification of the cell cycle kinase TLK2 presents an attractive genomic target for aggressive ER-positive breast cancers.

Amplification of TLK2 Induces Genomic Instability via Impairing the G2/M Checkpoint.

Managing aggressive breast cancers with enhanced chromosomal instability (CIN) is a significant challenge in clinics. Previously, we described that a cell cycle–associated kinase called Tousled-like kinase 2 (TLK2) is frequently deregulated by genomic amplifications in aggressive estrogen receptor–positive (ER+) breast cancers. In this study, it was discovered that TLK2 amplification and overexpression mechanistically impair Chk1/2-induced DNA damage checkpoint signaling, leading to a G2–M checkpoint defect, delayed DNA repair process, and increased CIN. In addition, TLK2 overexpression modestly sensitizes breast cancer cells to DNA-damaging agents, such as irradiation or doxorubicin. To our knowledge, this is the first report linking TLK2 function to CIN, in contrast to the function of its paralog TLK1 as a guardian of genome stability. This finding yields new insight into the deregulated DNA damage pathway and increased genomic instability in aggressive ER+ breast cancers. Implications: Targeting TLK2 presents an attractive therapeutic strategy for the TLK2-amplified breast cancers that possess enhanced genomic instability and aggressiveness. Mol Cancer Res; 14(10); 920–7. ©2016 AACR.

Abstract P1-07-07: Recurrent ESR1-CCDC170 rearrangements in an aggressive subset of estrogen-receptor positive breast cancers

The crucial role of gene fusions in epithelial tumorigenesis has been recently appreciated by several milestone discoveries, but no significant recurrent translocations have yet been found in the vast majority of breast cancers that express the estrogen receptor (ER). While a majority of ER+ tumors can be effectively treated by endocrine therapy, tumors of the luminal B subtype are more aggressive and endocrine therapy-resistant. Further, the molecular blueprint of these aggressive tumors is poorly understood. Thus, characterizing the genetic alterations underlying the more aggressive forms of ER+ breast cancer is of critical significance in breast cancer management. In this study, by large-scale analyses of breast cancer transcriptome and copy number alterations, we identified recurrent rearrangements between estrogen receptor gene, ESR1 and its neighbor gene, CCDC170 , which are enriched in the more aggressive and endocrine-resistant luminal-B tumors. Further screening of 200 ER+ breast tumor tissues by RT-PCR identified eight ESR1-CCDC170 positive tumors. The genomic rearrangements underlying these fusions were verified by genomic PCR and capillary sequencing. CCDC170 encodes a protein with unknown function. The observed fusion joins the 5’-untranslated region of ESR1 upstream to the coding region of CCDC170 , enabling the expression of N-terminally truncated CCDC170 (ΔCCDC170) under the promoter of the ESR1 gene. Consistent with the behavior of luminal B tumors, forced expression of ΔCCDC170 in ER+ breast cancer cells leads to markedly increased cell motility, invasiveness and anchorage-independent growth, and reduced endocrine sensitivity in vitro, as well as enhanced xenograft tumor formation and reduced endocrine sensitivity of the tumors in vivo. When introduced into benign breast epithelial cells, ΔCCDC170 impairs acini morphogenesis and enhances cell motility and invasiveness. Further, Knockdown of the endogenous ESR1-CCDC170 fusion variant expressed in HCC1428 cells potently inhibited cancer cell proliferation and migration, which can be rescued by forced expression of this fusion. Mechanistic studies suggest that ΔCCDC170 engages Gab1 signalosome to potentiate growth factor signaling and enhance cell motility. The augmented growth factor signaling driven by ΔCCDC170 appears to be sustained even after withdrawal of serum, and is not affected by endocrine treatment. Together, this study identified neoplastic ESR1-CCDC170 fusions in a more aggressive subset of ER+ breast cancer, which also suggests a new role of ER in breast tumorigenesis by contributing its promoter to an oncogene. Citation Format: Jamunarani Veeraraghavan, Ying Tan, Xi-Xi Cao, Jin-Ah Kim, Xian Wang, Dean P Edwards, Alejandro Contreras, Susan G Hilsenbeck, Eric C Chang, Rachel Schiff, Xiao-Song Wang. Recurrent ESR1-CCDC170 rearrangements in an aggressive subset of estrogen-receptor positive breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-07.

Abstract 3032: Genomic deregulation and therapeutic role of the cell-cycle kinase TLK2 in more aggressive breast cancers

More aggressive breast cancers often coincide with enhanced chromosomal instability (CIN), which poses a great challenge to clinical management. By integrative analysis of genomic data, we observed the amplification of tousled-like kinase 2 (TLK2), a cell cycle serine-threonine kinase, in approximately 9% of breast cancer, which is more frequent in the luminal B and late-stage breast tumors. The resulting TLK2 upregulation correlates with the level of CIN in breast tumors, and predicts worse outcome regardless of endocrine therapy. Further studies suggest that TLK2 overexpression leads to a G2/M checkpoint defect, delayed DNA repair, and enhanced invasiveness. TLK2 silencing selectively inhibits the growth of TLK2-high breast cancer cells, and induces apoptosis. This response is retained in the breast cancer cells with acquired endocrine resistance. TLK2 inhibition in a preclinical tumor model significantly improved progression-free survival. Together, targeting TLK2 presents an attractive therapeutic strategy on TLK2-amplified breast cancers. Citation Format: Jin-Ah KIM, Ying Tan, Xian Wang, Xixi Cao, Jamunarani Veeraraghavan, Yulong Liang, Dean P. Edwards, Xuewen Pan, Kaiyi Li, Rachel Schiff, Xiaosong Wang. Genomic deregulation and therapeutic role of the cell-cycle kinase TLK2 in more aggressive breast cancers. [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 3032. doi:10.1158/1538-7445.AM2015-3032