Models Of HER2-positive Breast Cancer Research Articles

The PTEN and ATM axis controls the G1/S cell cycle checkpoint and tumorigenesis in HER2-positive breast cancer

The tumor suppressor PTEN is disrupted in a large proportion of cancers, including in HER2-positive breast cancer, where its loss is associated with resistance to therapy. Upon genotoxic stress, ataxia telangiectasia mutated (ATM) is activated and phosphorylates PTEN on residue 398. To elucidate the physiological role of this molecular event, we generated and analyzed knock-in mice expressing a mutant form of PTEN that cannot be phosphorylated by ATM (PTEN-398A). This mutation accelerated tumorigenesis in a model of HER2-positive breast cancer. Mammary tumors in bi-transgenic mice carrying MMTV-neu and Pten398A were characterized by DNA damage accumulation but reduced apoptosis. Mechanistically, phosphorylation of PTEN at position 398 is essential for the proper activation of the S phase checkpoint controlled by the PI3K–p27Kip1–CDK2 axis. Moreover, we linked these defects to the impaired ability of the PTEN-398A protein to relocalize to the plasma membrane in response to genotoxic stress. Altogether, our results uncover a novel role for ATM-dependent PTEN phosphorylation in the control of genomic stability, cell cycle progression, and tumorigenesis.

AKT-mediated phosphorylation of Sox9 induces Sox10 transcription in a murine model of HER2-positive breast cancer

BackgroundApproximately 5–10% of HER2-positive breast cancers can be defined by low expression of the Ste20-like kinase, SLK, and high expression of SOX10. Our lab has observed that genetic deletion of SLK results in the induction of Sox10 and significantly accelerates tumor initiation in a HER2-induced mammary tumor model. However, the mechanism responsible for the induction of SOX10 gene expression in this context remains unknown.MethodsUsing tumor-derived cell lines from MMTV-Neu mice lacking SLK and biochemical approaches, we have characterized the signaling mechanisms and relevant DNA elements driving Sox10 expression.ResultsBiochemical and genetic analyses of the SOX10 regulatory region in SLK-deficient mammary tumor cells show that Sox10 expression is dependent on a novel −7kb enhancer that harbors three SoxE binding sites. ChIP analyses demonstrate that Sox9 is bound to those elements in vivo. Our data show that AKT can directly phosphorylate Sox9 in vitro at serine 181 and that AKT inhibition blocks Sox9 phosphorylation and Sox10 expression in SLK(-/-) tumor cells. AKT-mediated Sox9 phosphorylation increases its transcriptional activity on the Sox10 −7kb enhancer without altering its DNA-binding activity. Interestingly, analysis of murine and human mammary tumors reveals a direct correlation between the levels of active phospho-Sox9 S181 and Sox10 expression.ConclusionsOur results have identified a novel Sox10 enhancer and validated Sox9 as a direct target for AKT. As Sox10 is a biomarker for triple-negative breast cancers (TNBC), these findings might have major implications in the targeting and treatment of those cancers.

Antimetastatic and Antitumor Activities of Orally Administered NAX014 Compound in a Murine Model of HER2-Positive Breast Cancer.

The natural isoquinoline alkaloid Berberine (BBR) has been shown to possess several therapeutic effects, including anticancer activity. Different BBR derivatives have been designed and synthesized in order to obtain new compounds with enhanced anticancer efficacy. We previously showed that intraperitoneal (IP) administration of the BBR-derived NAX014 compound was able to counteract HER-2 overexpressing mammary tumors onset and progression in transgenic mice. However, the IP administration was found to induce organ toxicity at doses higher than 2.5 mg/Kg. In this study, we evaluated the effect of intragastric (IG) administration of 20 mg/kg of NAX014 on both safety and anticancer efficacy in HER-2/neu transgenic mice. Furthermore, cancer cell dissemination and migration, tumor cell senescence and immunological changes were examined. Our results demonstrated that IG NAX014 administration delayed the onset of mammary tumors with no negative effects on health and survival. NAX014 reduced HER-2 overexpressing BC cells migration in vitro and the frequency of lung metastasis in HER-2/neu transgenic mice. A statistically significant increase of senescence-associated p16 expression was observed in tumors from NAX014-treated mice, and the induction of cell senescence was observed in HER-2 overexpressing BC cells after in vitro treatment with NAX014. Although NAX014 did not modulate the presence of tumor-infiltrating lymphocytes, the level of circulating TNF-α and VEGF was found to be reduced in NAX014-treated mice. The overall results address the NAX014 compound as potential tool for therapeutic strategies against HER-2 overexpressing breast cancer.

Abstract PS10-08: Tucatinib potentiates the activity of the antibody-drug conjugate T-DM1 in preclinical models of HER2-positive breast cancer

Abstract Background: Tucatinib is an orally administered, reversible, highly specific HER2 tyrosine kinase inhibitor recently approved by the FDA in combination with trastuzumab and capecitabine for adult patients with advanced unresectable or metastatic HER2-positive breast cancer (mBC), including patients with brain metastases, that have failed at least one anti-HER2 regimen in the metastatic setting. In a phase IB clinical trial, tucatinib in combination with the HER2-targeted antibody-drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1) was well tolerated and demonstrated activity in heavily pre-pretreated patients with HER2-positive mBC (NCT01983501; Borges VF et al., 2018). We previously presented preclinical data that tucatinib increases the activity of trastuzumab-derived ADCs in HER2-positive breast cancer models. Here, we provide mechanistic insight that tucatinib potentiates the activity of T-DM1 by modulating HER2 protein dynamics and facilitating increased cytotoxic maytansinoid drug delivery. Methods: To assess changes to HER2 protein levels upon treatment with tucatinib, HER2-amplified breast cancer cell lines were analyzed by Western blot and quantitative FACS (qFACS). To probe the dynamics of HER2 at the cell surface upon binding to antibody therapeutics, SK-BR-3 cells were incubated with fluorescently labeled trastuzumab to mark HER2 at the cell surface. Cells were imaged over 72 hours to observe the internalization of surface-bound antibody. Concurrent experiments were conducted with trastuzumab labeled with QF01, a quenched fluor which fluoresces only upon lysosomal processing and can serve as a proxy for antibody catabolism. To directly measure the rates of ADC catabolism, lysates were generated from BT-474 cells treated with T-DM1 in the presence or absence of tucatinib over a 72 hour time course, and were analyzed by mass spectrometry for the T-DM1 adduct, Lys-MCC-DM1. Results: In HER2-amplified breast cancer cell lines, treatment with tucatinib increased overall and cell membrane-localized HER2 levels. As demonstrated by internalization assays, tucatinib had an initial effect that increased the dwell time of HER2 at the cell surface of SK-BR-3 cells. At later timepoints, HER2 bound to trastuzumab was internalized and directed towards lysosomes. These data were supported by parallel intracellular Lys-MCC-DM1 measurements, which demonstrated increased concentration of the adduct when TDM-1 was administered in combination with tucatinib. These data provide a mechanistic rationale as to why the co-administration of tucatinib with T-DM1 in vitro was synergistic by isobologram analysis, and why the combination of tucatinib with T-DM1 was more effective in vivo than either single agent alone in BT-474 xenografts and in PDX models tested, producing a higher proportion of partial or complete tumor regressions. Conclusions: The described preclinical in vitro and in vivo data of simultaneous dual HER2 inhibition with tucatinib and T-DM1, along with the results of the phase IB/2 clinical trial demonstrating preliminary safety and efficacy of the combination, warrant further clinical development of tucatinib in combination with T-DM1. These results also support the evaluation of tucatinib in combination with other HER2-targeted ADCs in patients with HER2-positive mBC. Citation Format: Anita Kulukian, Janelle Taylor, Nishi Jain, Devra Olson, Margo Zaval, Robert Thurman, Shawna Hengel, Lauren Farr, Thomas Pires, Scott R. Peterson. Tucatinib potentiates the activity of the antibody-drug conjugate T-DM1 in preclinical models of HER2-positive breast cancer [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 PS10-08.

Abstract PS10-06: Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib

Abstract Introduction: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified or mutated across various cancer types. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. All three TKIs bind and inhibit the kinase domain of HER2 but differ both in the mechanism of binding and in specificity for other HER family members. Direct comparisons to differentiate the pre-clinical efficacy of the three TKIs have been limited to small-scale studies and novel biomarkers of response to further define appropriate patient populations are required. Methods: In this study, the anti-proliferative effects of the three TKIs were compared using a 115-cancer cell line panel, including 12 breast cancer cell lines and 22 cell lines harbouring point mutations or amplifications of EGFR, HER2, or HER3. Hierarchical clustering analysis was carried out to compare the IC50 “fingerprint” of the three TKIs to 168 other anti-cancer agents. Novel markers of TKI sensitivity and resistance were identified through cross-analysis of each drug response profile with mutation, copy number variation, and gene expression data. Results: All three TKIs were effective against HER2-positive breast cancer models; neratinib showed the most potent activity, followed by tucatinib and lapatinib respectively (Table 1). Neratinib displayed the greatest anti-proliferative activity in HER2-mutant and EGFR-mutant cell lines. Clustering analysis revealed that the anti-proliferative profile of tucatinib was most similar to trastuzumab, while neratinib and lapatinib were most like other HER family inhibitors. Mutation and gene expression analysis identified potential markers of response for each TKI. High expression of four genes (HER2, VTCN1, CDK12, and RAC1) correlated with response to all three TKIs. DNA damage repair genes were significantly associated with resistance to the HER2-targeted TKIs. BRCA2 mutation was correlated with neratinib and tucatinib response, and high gene expression of ATM, BRCA2, and BRCA1 were all associated with neratinib resistance. Conclusions: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed possible mechanisms that may be exploited using combinatorial strategies involving CDK inhibitors, immunotherapies, and targeting DNA repair pathways. Table: IC50 values for neratinib, lapatinib, and tucatinib in the HER2+ breast cancer cell linesIC50 values (nM)Cell linesNeratinibLapatinibTucatinibAU-56520294125BT-4745926229HCC195413814262122MDA-MB-453306228445928SKBR3715222 Citation Format: Neil T Conlon, Jeffrey J Kooijman, Suzanne JC van Gerwen, Winfried R Mulder, Guido JR Zaman, Irmina Diala, Lisa D Eli, Alshad S Lalani, John Crown, Denis Collins. Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib [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 PS10-06.

Gut Microbiota Condition the Therapeutic Efficacy of Trastuzumab in HER2-Positive Breast Cancer.

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B-positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota β-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response. SIGNIFICANCE: Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients.See related commentary by Sharma, p. 1937 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2195/F1.large.jpg.

A Novel Proteomic Method Reveals NLS Tagging of T-DM1 Contravenes Classical Nuclear Transport in a Model of HER2-Positive Breast Cancer

The next breakthrough for protein therapeutics is effective intracellular delivery and accumulation within target cells. Nuclear localization signal (NLS)-tagged therapeutics have been hindered by the lack of efficient nuclear localization due to endosome entrapment. Although development of strategies for tagging therapeutics with technologies capable of increased membrane penetration has resulted in proportional increased potency, nonspecific membrane penetration limits target specificity and, hence, widespread clinical success. There is a long-standing idea that nuclear localization of NLS-tagged agents occurs exclusively via classical nuclear transport. In the present study, we modified the antibody-drug conjugate trastuzumab-emtansine (T-DM1) with a classical NLS linked to cholic acid (cell accumulator [Accum]) that enables modified antibodies to escape endosome entrapment and increase nuclear localization efficiency without abrogating receptor targeting. In parallel, we developed a proteomics-based method to evaluate nuclear transport. Accum-modified T-DM1 significantly enhanced cytotoxic efficacy in the human epidermal growth factor receptor 2 (HER2)-positive SKBR3 breast cancer system. We discovered that efficacy was dependent on the nonclassical importin-7. Our evaluation reveals that when multiple classical NLS tagging occurs, cationic charge build-up as opposed to sequence dominates and becomes a substrate for importin-7. This study results in an effective target cell-specific NLS therapeutic and a general approach to guide future NLS-based development initiatives.

Adipocyte-conditioned medium induces resistance of breast cancer cells to lapatinib

BackgroundThe existence of a cross-talk between peritumoral adipocytes and cancer cells has been increasingly investigated. Several studies have shown that these adipocytes protect tumor cells from the effect of anticancer agents.MethodsTo investigate a potential protective effect of adipocyte-conditioned medium on HER2 positive breast cancer cells exposed to tyrosine kinase inhibitors (TKI) such as lapatinib, we analyzed the sensitivity of HER2 positive breast cancer models in vitro and in vivo on SCID mice in the presence or absence of adipocytes or adipocyte-conditioned medium.ResultsConditioned medium from differentiated adipocytes reduced the in vitro sensitivity of the HER2+ cell lines BT474 and SKBR3 to TKI. Particularly, conditioned medium abrogated P27 induction in tumor cells by lapatinib but this was observed only when conditioned medium was present during exposure to lapatinib. In addition, resistance was induced with adipocytes derived from murine NIH3T3 or human hMAD cells but not with fibroblasts or preadipocytes. In vivo studies demonstrated that the contact of the tumors with adipose tissue reduced sensitivity to lapatinib. Soluble factors involved in this resistance were found to be thermolabile. Pharmacological modulation of lipolysis in adipocytes during preparation of conditioned media showed that various lipolysis inhibitors abolished the protective effect of conditioned media on tumor cells, suggesting a role for adipocyte lipolysis in the induction of resistance of tumor cells to TKI.ConclusionsOverall, our results suggest that contact of tumor cells with proximal adipose tissue induces resistance to anti HER2 small molecule inhibitors through the production of soluble thermolabile factors, and that this effect can be abrogated using lipolysis inhibitors.

Abstract 1911: HER2 L755S mutation is associated with acquired resistance to lapatinib and neratinib, and confers cross-resistance to tucatinib in HER2-positive breast cancer models

Abstract Despite the availability of potent HER-targeted agents, de novo and acquired resistance is common and continues to pose a major challenge, especially in the advanced setting. Amassing evidence point to the importance of HER2 mutations, including the most common HER2 L755S mutation, in mediating anti-HER2 resistance. The HER2 L755S mutation, in particular, is observed to be enriched in metastatic lesions compared to primary breast tumors. The need for effective therapy to treat tumors harboring HER2 mutations prevails. We have previously reported that acquired resistance to lapatinib (L)-containing treatments, mediated by HER2 L755S, could be overcome by the recently FDA-approved irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (N). While N has shown great promise in patients with HER2-mutant metastatic breast cancer, its efficacy is somewhat limited. More recently, tucatinib, a HER2-selective TKI, has been shown to be effective in HER2-positive (+) brain metastases. Its potency in the context of HER2 mutations, however, has not yet been fully studied. In this study, we used the HER2+ BT474-L resistant (LR) cells, harboring endogenous HER2 L755S mutation, and parental (P) cells to first determine whether tucatinib may be effective in overcoming resistance mediated by HER2 mutations. Our results showed that while N effectively inhibited the growth of LR cells, although at a dose higher than that needed to inhibit the growth of naïve P cells, tucatinib failed to inhibit the growth of LR cells. Our results suggest that HER2 L755S mutation may confer cross-resistance to tucatinib. To further study mechanisms of resistance to 2nd generation anti-HER2 agents, we recently developed cell models with acquired resistance to N, through long-term exposure of the BT474-P and LR cells to increasing doses of N. These cells were profiled by reverse phase protein array (RPPA) and western blot analysis, which revealed restoration of HER2 phosphorylation in the NR derivatives, despite being cultured in the continuous presence of N. Interestingly, RNA-seq analysis revealed the presence of HER2 L755S mutation in all the NR derivatives, but not in the P cells, suggesting that the reactivated HER2 signaling observed in NR cells could be attributed to the emergence/acquisition of HER2 L755S mutation. Furthermore, while the P cells were highly sensitive to tucatinib, L, and the monoclonal antibody trastuzumab (T), the NR derivatives were totally resistant to these agents, suggesting that N resistance may also confer cross-resistance to tucatinib, L, and T. Additional molecular characterization to examine differential gene expression and mutational profile of the resistant derivatives, as well as testing of novel anti-HER2 regimens and drug combinations targeting downstream mediators to overcome resistance, both in vitro and in vivo, is ongoing. Citation Format: Jamunarani Veeraraghavan, Ragini Mistry, Sarmistha Nanda, Vidyalakshmi Sethunath, Martin Shea, Tamika Mitchell, Meenakshi Anurag, Michael A. Mancini, Fabio Stossi, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. HER2 L755S mutation is associated with acquired resistance to lapatinib and neratinib, and confers cross-resistance to tucatinib in HER2-positive breast cancer models [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 1911.

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2\u2009+\u2009murine breast cancer model

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity.

Imaging and monitoring HER2 expression in breast cancer during trastuzumab therapy with a peptide probe 99mTc-HYNIC-H10F.

The novel molecular imaging probe 99mTc-HYNIC-H10F was developed for patient screening and efficacy monitoring of trastuzumab therapy by SPECT imaging of HER2 expression in breast cancer. 99mTc-HYNIC-H10F was developed by labeling H10F peptide with 99mTc following an optimized protocol. Biodistribution and SPECT/CT were performed in mouse models bearing HER2-positive SK-BR3 and HER2-negative MDA-MB-231 human breast cancer xenografts, respectively. The treatment response to trastuzumab was monitored and quantified by SPECT/CT in two HER2-positive breast cancer models (SK-BR3 and MDA-MB-361). The preliminary clinical study was performed in two patients with breast cancer. SPECT/CT with 99mTc-HYNIC-H10F showed that the SK-BR3 tumors were clearly visualized, while the signals from MDA-MB-231 tumors were much lower. The tumor uptake of 99mTc-HYNIC-H10F could be blocked by excess unlabeled H10F peptide but not by excess trastuzumab. The growth of two HER2-positive tumors was prominently suppressed at day 11 post-treatment. However, SPECT/CT reflected much earlier therapy response at day 4 post-treatment. The HER2 expression in tumors of breast cancer patients could be detected by 99mTc-HYNIC-H10F SPECT/CT imaging. 99mTc-HYNIC-H10F specifically accumulates in HER2-positive tumors. Compared with trastuzumab, 99mTc-HYNIC-H10F binds to a different domain of HER2 antigen, providing new opportunities to monitor HER2 expression levels before/during/after trastuzumab treatment for more effective personalized treatment.

Abstract P2-04-05: Loss of Ste20-like kinase induces a basal/stem-like phenotype in HER2-positive breast cancers

Abstract Over the past two decades, the mammalian Ste20-like kinase (SLK) has been characterized for its role in regulating cellular migration, proliferation and apoptosis in fibroblasts and myoblasts. In mammary epithelial cells, SLK has been shown to be required for efficient epithelial-to-mesenchymal transition and to be activated downstream of the HER2/Neu-oncogene to control chemotactic cellular migration. Here, we assessed the role of SLK in HER2/Neu-induced mammary tumorigenesis in vivo. As SLK is activated downstream of HER2/Neu, we hypothesized that the loss of SLK would significantly delay tumor progression in a mouse model of HER2-positive breast cancer. As we have shown that global attenuation of SLK kinase activity results in embryonic lethality, a conditional SLK knockout mouse model was generated. To study the role of SLK in HER2-positive breast cancer, we crossed these conditional SLK knockout mice with mice expressing HER2/Neu linked to Cre recombinase in the mammary luminal epithelium. Unexpectedly, we have demonstrated that conditional deletion of SLK significantly accelerates Neu-induced mammary tumor onset and decreases overall survival. SLK deletion results in the induction of Sox10 which drives mammary stem/progenitor activity and cooperates with HER2/Neu to drive tumor growth. Using the Cancer Genome Atlas, we have supported previous findings and validated Sox10 as a potential biomarker of the Triple-negative Breast Cancer subtype. Furthermore, we have uncovered that SLK deletion results in enhanced activation of AKT. We provide evidence that Sox10 induction requires signaling through a novel AKT/Sox9-dependent pathway following SLK deletion. Taken together, our data suggests that SLK may be required to maintain cells in a fully differentiated state and that loss of SLK in HER2/Neu-induced breast cancer drives a more basal/stem-like phenotype through the induction of Sox10. Citation Format: Khalid N Al-Zahrani, John Abou-Hamad, Ben Pryce, David P Cook, Johnathan Hodgins, Cedrik Labreche, Pascale Robineau-Charette, Christiano de Souza, John Bell, Rebecca Auer, Michele Ardolino, Barbara Vanderhyden, Daniel Schramek, Luc Sabourin. Loss of Ste20-like kinase induces a basal/stem-like phenotype in HER2-positive breast cancers [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 P2-04-05.

Abstract P4-14-11: Prolonged survival of mice bearing HER2 positive breast cancer with a therapeutic cancer vaccine via intra-reconstructed abdominal flap administration

Abstract Background: Previously we reported a porous silicon micro-particle based cancer vaccine, which stimulated robust CD8+ T cell-dependent anti-tumor immunity in mice bearing HER2-positive breast cancer. In this study we evaluated the efficacy of the vaccine via single injection into reconstructed abdominal flap after primary tumor mastectomy. Method: To generate murine model of HER2 positive breast cancer murine, 1 × 105 TUBO/luciferase cells were injectedinto both mammary gland fat pad and left ventricle of each 6-8 weeks old female Balb/c mice. Primary and systemic metastatic tumor growth were monitored with a IVIS-200 imaging system. Four days after tumor inoculation, mammary gland fat pad with primary tumor was resected by mastectomy, and superficial inferior epigastric(SIE) vessels based abdominal flap was used for abdominal reconstruction. During the surgery, mice also received one of the three treatments below: 1) negative control without treatments; 2) single intra-flap vaccination of cancer vaccine; and 3) single injection of cancer vaccine via footpad injection. Therapeutic efficacy were monitored with IVIS system weekly. To analyze immune response, popliteal and inguinal lymph nodes were collected one day and seven days post vaccination, and activation of dendritic cells and T cells was evaluated with a flow cytometry. HER2 specific T cells in splenocytes which highly expressed interferon-gamma (IFNγ) were counted by ELISpot. CD3+ T cell infiltration in brain metastatic nodules were analyzed by immunohistochemical assay. Result: Prolonged survival of mice bearing HER2-positive breast cancer was achieved by both intra-flap and intra-dermal vaccination of cancer vaccine. Mice vaccinated via intra-flap injection extended median survival by 9 days (p=0.025), although not as long as intra-dermal treatment (15 days, p=0.034), comparing to control group. In lymph nodes, activated dendritic cells increased in both two vaccinated groups. Interestingly, intra-flap vaccination induced higher response in inguinal lymph node, which considered as the major draining lymph node of this route. Meanwhile, intra-dermal vaccination raised higher immune response in popliteal lymph node. In ELISpot assay, IFNγ secreted T cells were found abundant in splenocytes from both two vaccination groups. Furthermore, efficient infiltration of T cells inside the brain metastatic nodules was found in intra-dermal injection group, which provided potential protection from distal recurrence after mastectomy. Conclusion: Cancer vaccine administrated via intra-flap injection effectively stimulated systemic immune response, and slowed down tumor progression in murine HER2 positive breast cancer model. Citation Format: Xiaoling Liu, Junhua Mai, Chaoyang Meng, Wei Wei, Haifa Shen. Prolonged survival of mice bearing HER2 positive breast cancer with a therapeutic cancer vaccine via intra-reconstructed abdominal flap administration [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 P4-14-11.

Abstract P6-03-20: The development and characterization of novel HER2-positive breast cancer models of acquired neratinib resistance

Abstract Introduction: Neratinib is an irreversible, small molecule pan-HER tyrosine kinase inhibitor that has shown clinical activity against HER2-amplified (HER2+) and HER2-mutated breast cancer. However, there are limited data on potential mechanisms of resistance to neratinib. This study aimed to develop HER2+ breast cancer cell line models of acquired neratinib resistance, and to elucidate the effects of acquired resistance on HER family signalling and drug response. As neratinib has activity in both estrogen receptor positive (ER+) and ER- HER2+ breast cancer, neratinib-resistant cell lines were generated and characterised from breast cancer cell lines of both subtypes. Methods: HER2+, ER- HCC1569 cells were continuously exposed to 150 nM neratinib for six months to generate the neratinib-resistant HCC1569 (HCC1569-N) cell line. HER2+ ER+ BT474 cells were continuously exposed to escalating doses of 5-80 nM neratinib for six months to generate the BT474-N cell line. Sensitivity to HER2-targeted therapies (neratinib, lapatinib and trastuzumab) and chemotherapeutics (5’dFUR, carboplatin, and vinorelbine) were assessed by 5-day acid phosphatase based proliferation assay. IC50 and combination index (CI) values were calculated using CalcuSyn software. Total and phosphorylated levels of HER2, EGFR, ERK, and Src were assessed by Western blotting following neratinib treatment (150 nM), to determine changes in HER2 signalling. Results: The HCC1569-N and BT474-N cells had a 50.3-fold and 105-fold increase in neratinib resistance. In addition, both cell lines developed cross-resistance to lapatinib and were resistant at clinically relevant concentrations (> 1 µM). HCC1569-Par cells are innately resistant to trastuzumab, which was not altered in HCC1569-N cells. However, neratinib resistance also caused resistance to trastuzumab in the BT474-N cell line, compared to its trastuzumab-sensitive parental cell line (Table 1). The HCC1569-N cell line did not show any significant difference in sensitivity to 5’dFUR, carboplatin, or vinorelbine compared to parental HCC1569, but the addition of vinorelbine to neratinib was synergistic (CI value = 0.28 ± 0.19). HCC1569-N cells displayed no changes in total protein or phosphorylated levels of HER2 or EGFR compared to parental cells. Additionally, neratinib treatment still resulted in inhibition of HER2, EGFR, and downstream ERK signalling, indicating a lack of reliance on HER2 signalling in HCC1569-N cells. Phosphorylated Src levels were elevated (p = 6.3 × 10−4) in HCC1569-N compared to parental cells. Conclusions: Neratinib resistance in HCC1569-N and BT474-N cells confers cross-resistance to other HER2-targeted therapies. Increased Src activity may be implicated in the neratinib-resistant HER2+ ER- phenotype. Cell line10 µg/mL Trastuzumab (%Growth)NeratinibIC50 valueLapatinibIC50 valueHCC1569-Par97.6 ± 2.57.8 ± 5.8653.0 ± 144.0HCC1569-N92.7 ± 2.9393.0 ± 77.1> 1000BT474-Par44.7 ± 1.81.2 ± 0.323.9 ± 19.6BT474-N97.8 ± 3.3126.8 ± 53.5> 1000 Citation Format: Neil T Conlon, Alacoque Browne, Laura Breen, Alex J Eustace, John Crown, Denis M Collins. The development and characterization of novel HER2-positive breast cancer models of acquired neratinib resistance [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 P6-03-20.

ARX788, a novel anti-HER2 antibody-drug conjugate, shows anti-tumor effects in preclinical models of trastuzumab emtansine-resistant HER2-positive breast cancer and gastric cancer.

The majority of HER2-positive breast or gastric cancers treated with T-DM1 eventually show resistance to this agent. We compared the effects of T-DM1 and ARX788, a novel anti-HER2 antibody-drug conjugate, on cell growth and apoptosis in HER2-positive breast cancer and gastric cancer cell lines sensitive to T-DM1, gastric cancer cell lines resistant to T-DM1, HER2-negative breast cancer cell lines, and T-DM1-resistant xenograft models. ARX788 was effective in T-DM1-resistant in vitro and in vivo models of HER2-positive breast cancer and gastric cancer. ARX788 showed a pronounced growth inhibitory effect on all five HER2-positive cell lines tested, of which two gastric cancer cell lines had acquired resistance to T-DM1. ARX788 evoked more apoptotic events compared to T-DM1. While JIMT-1 and RN-87 xenograft tumors progressed on T-DM1 treatment, all such tumors responded to ARX788, and four out of the six JIMT-1 tumors and nine out of the twelve RN-87 tumors disappeared during the ARX788 treatment. Mice treated with ARX788 survived longer than those treated with T-DM1. The data support evaluation of ARX788 in patients with HER2-positive breast cancer or gastric cancer including cancers that progress during T-DM1 therapy.

Calcium signaling affects migration and proliferation differently in individual cancer cells due to nifedipine treatment

Several papers have reported that calcium channel blocking drugs were associated with increased breast cancer risk and worsened prognosis. One of the most common signs of breast tumors is the presence of small deposits of calcium, known as microcalcifications. Therefore, we studied the effect of dihydropyridine nifedipine on selected calcium transport systems in MDA-MB-231 cells, originating from triple negative breast tumor and JIMT1 cells that represent a model of HER2-positive breast cancer, which possesses amplification of HER2 receptor, but cells do not response to HER2 inhibition treatment with trastuzumab. Also, we compared the effect of nifedipine on colorectal DLD1 and ovarian A2780 cancer cells. Both, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and type 1 sodium calcium exchanger (NCX1) were upregulated due to nifedipine in DLD1 and A2780 cells, but not in breast cancer MDA-MB-231 and JIMT1 cells. On contrary to MDA-MB-231 and JIMT1 cells, in DLD1 and A2780 cells nifedipine induced apoptosis in a concentration-dependent manner. After NCX1 silencing and subsequent treatment with nifedipine, proliferation was decreased in MDA-MB-231, increased in DLD1 cells, and not changed in JIMT1 cells. Silencing of IP3R1 revealed increase in proliferation in DLD1 and JIMT1 cells, but caused decrease in proliferation in MDA-MB-231 cell line after nifedipine treatment. Interestingly, after nifedipine treatment migration was not significantly affected in any of tested cell lines after NCX1 silencing. Due to IP3R1 silencing, significant decrease in migration occurred in MDA-MB-231 cells after nifedipine treatment, but not in other tested cells. These results support different function of the NCX1 and IP3R1 in the invasiveness of various cancer cells due to nifedipine treatment.

HO-1 drives autophagy as a mechanism of resistance against HER2-targeted therapies

PurposeTargeted therapies have resulted in major advances in the treatment of HER2-positive breast cancers. Despite this, up to 70% of patients will develop resistance to treatment within 2 years and new strategies for targeting resistant disease are needed.MethodsTo identify potential resistance mechanisms, we used the mouse MMTV-NIC-PTEN+/− spontaneous model of HER2-positive breast cancer and the pan-HER family kinase inhibitor sapatinib. Vehicle and sapatinib-treated tumors were evaluated by immunohistochemistry and proteomic analysis. In vitro studies were carried out to define the role of heme oxygenase 1 (HO-1) and autophagy in resistance to sapatinib and lapatinib, another pan-HER family kinase inhibitor.ResultsTreatment of tumor-bearing MMTV-NIC-PTEN+/− mice with sapatinib resulted in delayed tumor progression and increased survival. However, tumors eventually progressed on treatment. Proteomic analysis identified proteins associated with cellular iron homeostasis as being upregulated in the sapatinib-treated tumors. This included HO-1 whose overexpression was confirmed by immunohistochemistry. Overexpression of HO-1 in HER2-expressing SKBR3 breast cancer cells resulted in reduced sensitivity to both pan-HER family kinase inhibitors sapatinib and lapatinib. This was associated with increased autophagy in the HO-1 over-expressing cells. Furthermore, increased autophagy was also seen in the sapatinib-treated tumors. Treatment with autophagy inhibitors was able to increase the sensitivity of the HO-1 over-expressing cells to both lapatinib and sapatinib.ConclusionTogether these data indicate a role for HO-1-induced autophagy in resistance to pan-HER family kinase inhibitors.

Retinoid X receptor agonist LG100268 modulates the immune microenvironment in preclinical breast cancer models

Despite numerous therapeutic advances in the past decade, breast cancer is expected to cause over 42,000 deaths in the United States in 2019. Breast cancer had been considered an immunologically silent tumor; however recent findings suggest that immune cells play important roles in tumor growth even in the breast. Retinoid X receptors (RXRs) are a subclass of nuclear receptors that act as ligand-dependent transcription factors that regulate a variety of cellular processes including proliferation and differentiation; in addition, they are essential for macrophage biology. Rexinoids are synthetic molecules that bind and activate RXRs. Bexarotene is the only rexinoid approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Other more-potent rexinoids have been synthesized, such as LG100268 (LG268). Here, we report that treatment with LG 268, but not bexarotene, decreased infiltration of myeloid-derived suppressor cells and CD206-expressing macrophages, increased the expression of PD-L1 by 50%, and increased the ratio of CD8/CD4, CD25 T cells, which correlates with increased cytotoxic activity of CD8 T cells in tumors of MMTV-Neu mice (a model of HER2-positive breast cancer). In the MMTV-PyMT murine model of triple negative breast cancer, LG268 treatment of established tumors prolonged survival, and in combination with anti-PD-L1 antibodies, significantly (p = 0.05) increased the infiltration of cytotoxic CD8 T cells and apoptosis. Collectively, these data suggest that the use of LG268, a RXR agonist, can improve response to immune checkpoint blockade in HER2+ or triple-negative breast cancer.

64P - The potential of neratinib plus dasatinib in overcoming and preventing neratinib resistance in HER2-positive breast cancer models

BackgroundNeratinib (NER) is an irreversible pan-HER kinase inhibitor with demonstrated clinical activity for HER2-positive and HER2-mutated breast cancers (BC). Mechanisms of resistance to NER are poorly understood. The Src/Abl inhibitor dasatinib (DAS) has shown ability to overcome resistance to HER2-targeted agents such as lapatinib (LAP) and TRAS in vitro. This pre-clinical study investigated the efficacy of DAS in combination with NER to overcome or prevent NER resistance in BC models. MethodsAnti-proliferative effects of NER, LAP, TRAS, DAS, and NER plus DAS were assessed in five NER-resistant HER2+ BC cell lines (HCC1954-N, HCC1569-N, EFM192A-N, BT474-N, and SKBR3-N) by acid phosphatase assay. IC50values and Combination index (CI) values were calculated to determine synergy (CI<0.8) using Calcusyn. Neratinib resistance was defined as an IC50value > 150nM NER. Apoptosis induction was assessed by Caspase 3/7-Glo assay. Reverse phase protein array was used to determine changes in key signalling pathways in HCC1954-N cells. BC cells were treated twice weekly with NER and/or DAS and crystal violet stained when confluent to examine resistance development. ResultsAll NER resistant cell lines examined had significantly reduced response to NER (11-83 fold increase in IC50values versus parental cells), as well as LAP and TRAS, compared to their parental cells.The combination of NER and DAS displayed synergy in all five NER resistant cell lines (CI=0.1-0.6). NER plus DAS caused a strong induction of apoptosis in the HCC1954-N cells (p=0.015). NER/DAS treatment caused changes in 23 phospho- or total protein levels, including suppression of Akt, MAPK, Src, p38 and AMPK signalling. NER alone (9 phospho- and 1 total proteins) and DAS alone (3 phospho- and 3 total proteins) altered fewer targets. The addition of DAS to NER prevented the emergence of NER resistance in parental HCC1954 and HCC1569 cells. ConclusionsThis study provides pre-clinical rationale for the combination of NER and DAS in NER-resistant HER2+ BC and shows that this combination warrants further investigation. Legal entity responsible for the studyThe authors. FundingPuma Biotechnology. DisclosureN. Conlon: Research grant/Funding (institution): Puma Biotechnology. J. Crown: Full/Part-time employment: OncoMark; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Research grant/Funding (institution): Eisai; Honoraria (self): Amgen; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Puma Biotechnology; Honoraria (self): Seattle Genetics; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Travel/Accommodation/Expenses: Pfizer; Honoraria (self), Advisory/Consultancy: Vertex; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution), Travel/Accommodation/Expenses: Roche; Honoraria (self), Travel/Accommodation/Expenses: MSD Oncology; Travel/Accommodation/Expenses: AstraZeneca; Travel/Accommodation/Expenses: Abbvie; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Boehringer Ingelheim; Honoraria (self), Speaker Bureau/Expert testimony: Genomic Health. D.M. Collins: Research grant/Funding (institution): Puma Biotechnology; Research grant/Funding (institution): Roche. All other authors have declared no conflicts of interest.

Enchancement of Toremifene Anti-Tumor Action by Metformin and Unusual Side Effect of Toremifene in Male Transgenic Mice with HER2-Positive Breast Tumor.

HER2-positive breast tumors are found in 25-30% of patients with breast cancer and are characterized by aggressive course and reduced sensitivity to both chemotherapy and hormone therapy. The aim of the work was to study the possibilities of enhancing the therapeutic effect of anti-estrogen drug toremifene by combining it with biguanide, metformin, on the HER2-positive breast cancer model in FVB/N HER-2/neu transgenic mouse. Male FBV/N mice with intramuscularly transplanted HER2-positive mammary gland tumor from a female mouse of the same strain have been given toremifene (30 mg/kg, orally daily) or metformin (100 mg/kg, orally daily) that had a moderate antitumor effect (decrease the area under the kinetic curve of tumor growth by 1.6 and 1.5 times, respectively, when compared with intact control). Co-administration of these drugs in the same doses had a more pronounced effect (the area under the kinetic curve of tumor growth decreased by 3.1 times compared to intact control; p<0.05). After 10 days, in group receiving toremifene all 10 mice developed inguinal-scrotal hernias, and in group that received toremifene plus metformin - only 5 of 10 (p=0.0325). By the 15th day after the start of treatment, the hernias was also determined in all mice treated with the combination of toremifene and metformin, but the size of the hernial sac was significantly smaller than in those receiving only toremifene - 537 ± 96 mm3 and 1309 ± 120 mm3, respectively (p=0.0001). A possible explanation is the manifestation of collagen-degrading effect of toremifene.