Development of immunocompetent models for primary and metastatic ER+ breast cancer.

Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Background.Targeting HER2 gene amplification is one of the great achievements in oncology resulting in the use of a wide array of anti-HER2 agents in the clinic. Unfortunately, 20% of patients still relapse with secondary organ metastasis and are currently incurable. While only 1.6% of primary non-HER2-amplified ER+ breast cancers harbor HER2 mutations, 6-10% of all metastatic breast cancers harbor HER2 mutations, suggesting their causal role in contributing to metastasis. The clinical value of HER2 activating mutations is being tested with the pan-HER tyrosine kinase inhibitor neratinib in phase II clinical trials (NCT01670877, NCT01953926, and NCT02465060). To date, neratinib has elicited only modest responses in ER+ breast cancer, often rapidly followed by progression. This study characterizes HER2-mutant induced resistance to endocrine therapy or neratinib induced resistance and resulting metastasis, in order to determine a more effective rational therapeutic approach for treating ER+ HER2-mutant breast cancer. Methods.HER2 mutation frequency and its impact on patient outcome was determined using METABRIC primary breast cancer (BC) and MSK IMPACT metastatic BC ER+ sequencing studies. Effects of estrogen (E2), fulvestrant, or neratinib on cell growth and HER2 signaling were examined on ER+/ILC cells (MM134) stably expressing HER2/WT, HER2/S310F, and HER2/L755S. Cell growth was measured using CellTiter-Glo and HER2 signaling was analyzed by western blot analysis. Additionally, the effect of these ER+/ILC and IDC HER2 mutations on tumor growth and endocrine or neratinib treatment resistance was determined using fat pad injections and MIND xenografts in NOD-scid gamma mice. Results.We searched ER+ sequencing datasets and identified HER2 mutations that are highly enriched in ER+ ILC as compared to ER+ IDC. These activating HER2 mutations in ER+ ILC are associated with early relapse and poor overall survival. Moreover, we are finding that ILC patients harboring the recurrent HER2/L755S mutation have worse overall survival compared to non-mutant HER2 ILC. MM134 cells expressing HER2/S310F and HER2/L755S show increased cell growth, strongly activated autophosphorylation of HER2, and increased downstream signaling (pMAPK and pAKT) as compared to cells expressing HER2/WT upon treatment with fulvestrant (1μM). Three clinically relevant in vivo models including ILC HER2/L755S Mammary INtraDuctal (MIND) xenografts, IDC HER2/L755S fat pad xenografts, and IDC HCI-003 (an ER+ patient-derived xenograft (PDX) naturally harboring the exon 20 activating HER2G778_P780 dup) exhibit fulvestrant and neratinib resistance and lung and ovary metastases. In addition, we find that HER2 mutations induced mTOR signaling. In contrast, however, the pan-HER drug poziotinib does potently inhibit tumor growth and organ-specific metastasis and perturbs mTOR activation in these models. Conclusion.We demonstrate that clinically associated HER2 mutations drive endocrine therapy or neratinib resistance and poor patient outcome in ER+ patients. Our data propose the use of the irreversible pan-HER TKI poziotinib for treating endocrine therapy or neratinib refractory ER+ HER2-mutant metastatic breast cancer. Citation Format: Shyam. M Kavuri, Rashi Kalra, Ching Hui Chen, Junkai Wang, Ahmad Bin Salam, Lacey Dobrolecki, Alaina Lewis, Christina Sallas, Clayton Yates, Carolina Gutierrez, Balasubramanyam Karanam, Meenakshi Anurag, Bora Lim, Matthew Ellis. Distinct HER2 allele specific therapeutic response and preclinical efficacy of poziotinib in metastatic ER+ HER2 mutant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-24.

Discovery and development of novel therapies in advanced breast cancer: rapid development of ribociclib

Override of the Osteoclast Defect in Osteopontin-Deficient Mice by Metastatic Tumor Growth in the Bone

Opportunities to prevent late-stage breast cancer within the course of usual care are needed. We evaluate whether clinical encounters offer such opportunities. Within seven health care plans, we identified 1298 women aged more than 50 years with early (<3 cm), late-stage (> or = 3 cm), or metastatic invasive breast cancer diagnosed during 1995-1999, whose first screening mammogram 13-36 months prior to the diagnosis (index) was negative. We audited all care occurring in the health plans up to 36 months prior to the cancer diagnoses. Ordinal logistic regression compared the frequency of events by disease category. We hypothesized that during the 13-36 months prior to diagnosis, women with late-stage or metastatic breast cancer would have more symptoms and be more likely to have breast-related clinical visits but have less breast screening (clinical breast examination [CBE] or mammography) than women with early-stage disease, thereby indicating clinical opportunities for earlier detection. We found no differences in demographic characteristics across breast cancer stage among the 1298 women. Both before and after the negative index mammogram but during the 13-36 months prior to diagnosis, few women had breast symptoms (5% before index, 8% after), but many sought breast care (86% before index, 90% after) and screening CBE (62% before index, 43% after). Only the occurrence of screening CBE (odds ratio [OR] = 0.73, 95% confidence interval [CI] = 0.56 to 0.95) or screening mammograms (OR = 0.74, 95% CI = 0.57 to 0.97) after the negative index mammogram reduced odds of more severe disease at diagnosis. Although the mortality benefit of CBE, or one compared to two year mammography has not been established, we found that women with late-stage breast cancers undetected by screening mammography did not experience opportunities for earlier detection except through CBE or additional screening mammography.

3092 Background: Breast cancers are most frequently oestrogen receptor (ER) and progesterone receptor (PR) positive and 18F-Fluorodeoxyglucose PET-CT (FDG) used in conventional staging of breast cancer has lower sensitivity for these subtypes. Gastrin releasing peptide receptors (GRPR) are a potential alternative diagnostic and theranostic target for ER+/PR+ breast cancers due to their overexpression of GRPR. This phase 1 study aims to assess the safety and potential of the novel radiotracer 64Cu-Sarcophagine(SAR)-Bombesin (BBN) in the re-staging of recurrent metastatic ER+/PR+/human epidermal growth factor 2 negative (HER2-) breast cancer. Methods: Patients with confirmed recurrent or primary metastatic ER+/PR+/HER2- breast cancer undergoing staging prior to a new treatment underwent 64Cu-SAR-BBN PET-CT with imaging at 1, 3 and 24 hours post-injection. Bloods and vital signs were acquired for patients at baseline, 1, 3 and 24 hours post-injection timepoints, and electrocardiogram (ECG) performed 1 hour pre and 1 hour post injection. Blood tracer-clearance and dosimetry was performed. GRPR receptor status was assessed in 4/7 patients from metastatic-site biopsy samples. Staging of the patients was assessed by conventional imaging (FDG, bone scan and diagnostic CT) within 3 weeks of 64Cu-SAR-BBN imaging. All PET scans were assessed visually, and quantitatively using MIM Software. Results: 9 patients were enrolled. 7/9 patients underwent all imaging modalities, while 2/9 did not undergo BBN imaging. 1/7 patient who underwent all imaging had de- novo metastatic ER+/PR+/Her 2- breast cancer and 6/7 recurrent metastatic disease. 2/7 had lobular subtype. There were no adverse events reported, and ECG, vitals and haematological, biochemical and coagulation markers remained unchanged. All 7 patients were positive on conventional imaging, while 6/7 were positive on FDG. BBN was positive in 5/7 patients. Both BBN negative patients had disease identified on FDG. Conversely, 1 patient was BBN positive but FDG negative. 4/7 patients were BBN positive and FDG positive. In these 4 patients, mean SUVmax was higher for BBN than FDG (15 vs. 12). In classical lobular subtype (2/7), BBN was highly avid compared to FDG (SUV max 20 vs 11, and 20 vs &lt;3) and with a higher tumor volume compared to FDG (2034 vs 504, and 634 mL vs FDG negative). Conclusions: 64Cu-SAR-Bombesin is a novel tracer which appears safe and may have a diagnostic and theranostic role in patients with metastatic ER+/PR+/HER2- breast cancer, particularly lobular subtype. Further evaluation appears warranted.

ABSTRACTIntroduction: For patients with late-stage (metastatic) breast cancer, the impact of treatment on health-related quality of life is a key factor in decision-making. A systematic review was conducted to identify health state utility values (HSUVs) for late-stage breast cancer, derived using methods preferred by health technology assessment (HTA) agencies, by treatment line. The aim was to generate a list of HSUVs, that could help to justify the values used to populate cost-utility models.Areas covered: Ten electronic databases, international congress websites and online HSUV databases were searched (January 1995–May 2014) for HSUVs for adults with late-stage breast cancer that had been derived from methods favoured by HTA agencies. Publications were included only if they reported studies that originated HSUVs.Expert commentary: Large numbers of HSUVs are available for late-stage breast cancer in the published literature. Contrary to expectations, the HSUVs reported in the literature vary greatly for some health states. As a result, the choice of HSUV can have considerable implications for the outcomes of economic evaluations. Standardization of HSUV methodology is expected to reduce variability; however, further research is recommended for assessing the sensitivity of generic preference-based measures in late-stage (metastatic) breast cancer.

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Background: The distinction of primary lung cancer from metastatic breast cancer is crucial in patients presenting with a solitary pulmonary nodule after breast surgery. However definitive diagnosis of these nodules is often difficult due to similar radiological and pathological features in primary lung and metastatic breast cancer nodules. We assessed the feasibility of our diagnostic approach for these nodules by morphopathological and immunohistochemical examination, and estimated the frequency of primary lung cancer occurrence in breast cancer patients. Material and Methods: We evaluated solitary pulmonary nodules appearing in 24 patients (0.62% : 24/3851) after breast surgery between 1994 and 2006. Patients with metastases to organs other than lungs were not included. For histological examination, CT-guided core needle biopsy (CT-CNB), trans-bronchial lung biopsy (TBLB), or surgical resection was performed. Besides conventional morphopathological examination using HE staining, differential diagnosis was performed by immunohistochemical examination (thyroid transcription factor-1: TTF-1, surfactant pro-protein B: SPPB, estrogen receptor: ER, mammaglobin1: MGB1). The immunopositive results for TTF-1 and SPPB support the specimen diagnosis of primary lung cancer. The immunopositive and immunonegative results for ER and both TTF-1 and SPPB, respectively, suggest that the specimen has a high probability of being metastatic breast cancer. And for cases in which differential diagnosis failed in the morpopathological and TTF-1, SPPB, and ER examination, MGB1 examination was conducted. Several patient and tumor characteristics were evaluated according to the definitive diagnosis of pulmonary nodules in both metastatic breast and primary lung cancer groups, which were then compared using Student's t-test. Results: Biopsy specimens were obtained using minimally invasive methods (CT-CNB and TBLB) in 21 patients (87.5%). Surgical resection was performed for diagnosis and treatment in three patients. Differential diagnosis was obtained by morphopathological methods alone in 18 patients (75.0%, primary lung cancer: 6 cases, metastaic breast cancer: 12 cases) and by immunohistochemical examination in the remaining 6 (25.0%, primary lung cancer: 1 case, metastaic breast cancer: 5 cases). Final diagnosis was metastatic breast and primary lung cancer in 17 (70. 8%) and 7 patients (29.2%), respectively. The mean age was significantly higher in the primary lung than metastatic breast cancer patients group (67 years vs. 57 years: p=0.036). No significant difference in the average diameter of pulmonary nodules was observed between metastatic breast and primary lung cancer patients (2.32cm vs. 2.29 cm). Furthermore, there were no clinical findings between metastatic breast cancer patients group and primary lung cancer group. Conclusions: Our results show the clinical feasibility of our approach to the differential diagnosis of breast cancer relapse and primary lung cancer presenting as a solitary nodule in patients after breast surgery. Further, replication of our findings under independent setting is recommended. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-03-05.

Palbociclib: A new breakthrough for advanced breast cancer

A long standing problem in anti-cancer drug development has been the limited value of preclinical mouse tumor models to reliably predict subsequent clinical activity. All too often highly encouraging preclinical results in mice are followed by complete failure in clinical trials, especially at the randomized phase III level. There are many possible reasons that have been postulated for this preclinical/clinical discrepancy. One which we have been studying for the past decade is the failure to use mouse models which duplicate the challenging circumstance of treating advanced (established) visceral metastatic disease after primary tumors have been surgically resected. Instead, preclinical treatment of established primary tumors or low volume (micro)metastatic disease, often confined to the lungs, have been the historical preclinical model norms. To address this problem we have developed several models of postsurgical advanced metastatic disease involving human tumor xenografts grown in SCID mice, including breast, colorectal or kidney cancer, and melanoma (1). This approach has been extended more recently for postsurgical adjuvant therapy of early stage microscopic metastatic disease (2,3). The established cell lines used for in vivo studies are variants previously selected in vivo for more aggressive spontaneous metastatic capability, which then are sometimes stably tagged with luciferase to permit whole body bioluminescent imaging. Using these models to evaluate the impact of several anti-cancer treatments, consisting mostly of antiangiogenic drugs and/or chemotherapy, either standard maximum tolerated dose or low-dose ‘metronomic’, has highlighted the critical contribution of the extent of metastatic disease to differential therapeutic outcomes, and the prospect of better correlation with clinical outcomes. For example, primary orthotopic tumors, e.g. breast cancer in the mammary fat pad, respond well to treatment with an antiangiogenic drug such as sunitinib, pazopanib or anti-VEGFR-2 antibodies whereas mice with advanced metastases in sites such as the liver or lungs do not, e.g. no prolongation of survival is observed (4). Adding chemotherapy, e.g. paclitaxel to sunitinib did not change the results, whereas adding DC101, an anti-VEGFR-2 antibody, to the same chemotherapy regimen did result in a modest survival improvement (thus mimicking the metastatic breast cancer E2100 phase III results of bevacizumab plus paclitaxel chemotherapy) (4). The observed lack of sunitinib efficacy alone or with chemotherapy when treating mice with advanced metastases mimics three failed phase III clinical trial results of this drug with or without chemotherapy in metastatic breast cancer patients (5). In addition, we have noted that successful treatment of mice with advanced systemic metastatic breast, melanoma or renal cell cancer, e.g. with low-dose metronomic chemotherapy plus an antiangiogenic drug such that overall survival is meaningfully prolonged, sometimes results in the emergence of overt spontaneous brain metastases(1,6,7). Thus, in mice, the brain appears to be a protective sanctuary for the survival and progressive growth of microscopic into macroscopic metastases, as already well known in the clinic. More recent studies have indicated how the brain microenvironment can contribute to the development of melanoma metastases in this organ environment e.g. the interaction of endothelins (ETs) with (elevated) endothelin receptor B expression by the brain melanoma metastatic variants (8). In summary, models of postsurgical advanced metastatic disease to undertake experimental therapeutic studies appear to have a greater degree of clinical relevance compared to most conventional primary tumor therapy models. We are now extending this approach to the development of postsurgical models of early stage microscopic metastatic disease to mimic adjuvant therapy in the clinic (2,9,10); some of our previous (2009) results indicated that adjuvant antiangiogenic therapy may actually worsen eventual survival outcomes of mice with early stage disease (2), a finding for which there is now some preliminary clinical support based on a recent phase III clinical trial assessing treatment of postsurgical early stage colorectal cancer patients with bevacizumab plus chemotherapy (11,12).

Background: Breast cancer (BC) is treatable with early detection, but once metastasis occurs, there is no cure. Therefore, it is of upmost importance to find targetable biomarkers that are involved in modulating the multi-step metastatic process. The Receptor for Advanced Glycation Endproducts (RAGE) and its ligands are an inflammatory pathway that are involved in modulating breast cancer progression and metastasis. We recently demonstrated that genetic or pharmacological interruption of RAGE signaling affected tumor progression and metastasis. However, no studies have dissected the role of RAGE in tumor growth from the metastatic cascade. Here, we show for the first time in multiple metastatic breast cancer models that targeting RAGE impairs BC metastasis. Methods: We tested the anti-metastatic effect of RAGE in vitro using the RAGE inhibitor FPS-ZM1 or RAGE shRNA knockdown in cell invasion, proliferation, migration and sphere formation assays (with 4T1, Py8119 and E0771 mouse BC cells). For in vivo assays we used orthotopic BC models (4T1/BALB/c and E0771 &amp; Py8119/C57BL6) and experimental metastasis assays (tail vein injection of 4T1/BALB/c and Py8119/C57BL6). To target RAGE, we used genetic (shRNA in 4T1 cells and RAGE knockout in C57BL6 mice) and pharmacological approaches (I.P. injection of FPS-ZM1). To investigate the synergistic effects of RAGE inhibition on progression and metastasis, we tested combination therapy of low-dose doxorubicin and FPS-ZM1. Results: Inhibition of RAGE with FPS-ZM1 (1-5uM) impaired tumor cell invasion of 4T1, E0771 and Py8119 cells. Similarly, in spheroid assays, treatment of cells with FPS-ZM1 resulted in fewer and smaller colonies. However, FPS-ZM1 treatment did not affect cell proliferation. In vivo studies revealed that FPS-ZM1 (1mg/kg) has a modest effect on tumor growth in 4T1/BALB/c injected mice but displayed a dramatic inhibitory effect on metastasis to the lungs. In experimental metastasis assays, tail-vein injection of 4T1 cells in BALB/c mice demonstrated that FPS-ZM1 treatment strongly impaired metastatic disease in mice compared to controls. To dissect the genetic role of RAGE in the tumor versus host, we test the effect of RAGE knockdown in 4T1 cells in experimental metastasis assays. RAGE shRNA impaired metastasis to the lungs, albeit to a lower degree seen with the RAGE inhibitor. To test the role of the host, we injected Py8119 cells into wild-type and RAGE knockout (RKO) mice. RKO mice displayed fewer metastatic burden compared to wild-type mice. Finally, in our combination treatment experiments, treatment of 4T1-injected BALB/c mice with Doxorubicin and FPS-ZM1 (alone and in combination), demonstrated that drug combination was more effective in inhibiting lung metastasis than either reagent alone. We are currently assessing how RAGE mechanistically drives these metastatic changes. Conclusion: Our data strongly suggests RAGE plays an important role in breast cancer metastasis, with less of an effect on tumor growth. Ongoing studies in our lab are testing which stage of the metastatic cascade RAGE is involved in, and the underlying mechanisms driving these processes. In conclusion, the use of RAGE inhibitors could represent a novel therapeutic approach for metastatic breast cancer. Citation Format: Gyong Ha Hwang, Melinda Magna, Barbara Mera, Toni Yeasky, Taekyoung Kwak, Lucas Outcault, Masaru Takabatake, Thuy-Mai Le, Marc E. Lippman, Barry I. Hudson. Targeting RAGE inhibits breast cancer invasion and metastasis [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 P3-01-11.

The phosphatidylinositol-3-kinase (PI3K) pathway is mutated and aberrantly activated in breast and other cancers and plays a key role in cancer cell proliferation and survival.1 2 The PI3K pathway is deregulated through a variety of mechanisms, including mutation or amplification of PI3K, loss or inactivation of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 ( PTEN ), as well as activation of tyrosine kinase growth factor receptors or oncogenes upstream of PI3K.3 4 Activating mutations in PIK3CA , the gene encoding the alpha isoform (p110 α) catalytic subunit of PI3K, is present in up to 40% of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers5 6 and represents a molecular target to personalise therapy of selected patients with breast cancer.2 Standard of care therapy for advanced HR-positive/HER2-negative breast cancers consists on endocrine therapy with or without the use of cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors.7 Therapy-resistance inevitably occurs in the majority of patients. The rationale of combining PI3K inhibitors and endocrine therapy is to synergistically inhibit both the PI3K and ER pathways.8 Initial trials of pan-PI3K inhibitors plus endocrine therapy for patients with advanced breast cancer showed modest benefit with high rates of toxicity limiting their clinical drug development.9–11 Selective isoform-specific PI3K inhibitors, such as an α-specific PI3K inhibitor, have subsequently revealed activity with less toxicity.12 13 The phase III SOLAR-1 (Clinical Studies of Alpelisib in Breast Cancer 1) trial investigated the efficacy and safety of alpelisib, a α-specific class-I PI3Kinhibitor, plus fulvestrant versus placebo plus fulvestrant in patients with metastatic HR-positive/HER2-negative breast cancer who had received endocrine therapy beforehand.14 About 85.6% of patients …

e13121 Background: Metastatic breast cancer remains a major clinical challenge, characterized by poor prognosis and limited therapeutic options. The incidence of metastatic female breast cancer increased from 5.8 to 7.9 per 100,000 females from 2001 to 2021. The Hippo pathway effector YAP1 plays a critical role in driving tumor growth and metastasis in many cancers including breast but little is known about the gene-specific changes that occur when breast cancers have metastasized. This study aimed to investigate the extent of genomic alterations, including copy number alterations (CNAs) and mutations, in YAP1 and its downstream genes in metastatic breast cancer compared to nonmetastatic breast cancer. Methods: Genomic data for the YAP1 gene and its downstream target genes (CTGF, Cyr61, MCL-1, TOP2A, FOSL2, and ANKRD1) were obtained from cBioPortal. This included mutation data and CNA data, considering both amplifications and deletions as CNA events. The chi-square test was performed using GraphPad Prism 10 to compare the frequencies of mutations and CNAs between metastatic and non-metastatic breast cancer cases. Results: A total of 2390 samples of metastatic breast cancer and 9289 samples of non-metastatic breast cancer were analyzed for mutations. Among them, a significantly higher percentage of mutations in the YAP1 (P ≤ 0.0001), ANKRD1 (P ≤ 0.0001), MCL-1 (P ≤ 0.0001), and TOP2A (P ≤ 0.0001) genes was observed in metastatic breast cancer compared to non-metastatic breast cancer. Evaluating CNAs in 2197 cases in the metastatic breast cancer group compared to 9289 cases in the non-metastatic breast cancer group demonstrated a significantly higher frequency of CNAs in the YAP1 ( P ≤ 0.0001), Cyr61 (P ≤ 0.0001), CTGF ( P ≤ 0.0001), ANKRD1 ( P ≤ 0.0001), TOP2A (P ≤ 0.0001) and FOSL2 (P ≤ 0.0001) genes among metastatic breast cancer. Conclusions: This study revealed significant genomic alterations in the YAP1 gene and its downstream genes in metastatic breast cancer. These findings highlight the importance of the YAP1 pathway in breast cancer metastasis and suggest that targeting this pathway may have therapeutic potential for primary and metastatic breast cancer. Frequency of mutations and copy number alterations (CNAs) in metastatic vs. non-metastatic breast cancer. Gene Metastatic Breast Cancer Mutation Frequency (%) Non-Metastatic Breast Cancer Mutation Frequency (%) P-value Metastatic Breast Cancer CNA Frequency (%) Non-Metastatic Breast Cancer CNA Frequency (%) P-value YAP1 0.75 0.17 &lt;0.0001 5.1 1.18 &lt;0.0001 CTGF 0.13 0.18 0.781 5.42 0.65 &lt;0.0001 Cyr61 0.17 0.16 0.999 3.9 0.65 &lt;0.0001 MCL-1 0.63 0.11 &lt;0.0001 10.74 11.93 0.121 ANKRD1 1.17 0.09 &lt;0.0001 1.96 0.25 &lt;0.0001 TOP2A 0.96 0.18 &lt;0.0001 8.06 3.6 &lt;0.0001 FOSL2 0.17 0.1 0.317 3.64 0.62 &lt;0.0001

New treatment modalities are urgently needed to better manage advanced breast cancer. Combination therapies are usually more effective than monotherapy. In this context, the use of cyclic and acyclic O,N-acetals derivative compounds in combination with the suicide gef gene shown a potent anti-tumor activity and represent a new generation of anticancer agents. Here, we evaluate the use of the gef gene to promote and increase the anti-tumor effect of cyclic and acyclic O,N-acetals purine derivatives and elucidate their mechanisms of action. Among all compounds tested, those with a nitro group and a cyclic pattern structures (FC-30b2, FC-29c, and bozepinib) are the most benefited from the gef gene effect. These compounds, in combination with gef gene, were able to abolish tumor cell proliferation with a minimal dose leading to more effective and less toxic chemotherapy. The effect of this combined therapy is triggered by apoptosis induction which can be found deregulated in the later stage of breast cancer. Moreover, the combined therapy leads to an increase of cell post-apoptotic secondary necrosis that is able to promote the immunogenicity of cancer cells leading to a successful treatment. This data suggests that this novel combination therapy represents a promising candidate for breast cancer treatment.