Patient-specific signaling signatures predict optimal therapeutic combinations for triple negative breast cancer.

Triple negative breast cancer (TNBC) is a heterogeneous group of tumors which lack estrogen receptor, progesterone receptor, and HER2 expression. Targeted therapies have limited success in treating TNBC, thus a strategy enabling effective targeted combinations is an unmet need. To tackle these challenges and discover individualized targeted combination therapies for TNBC, we integrated phosphoproteomic analysis of altered signaling networks with patient-specific signaling signature (PaSSS) analysis using an information-theoretic, thermodynamic-based approach. Using this method on a large number of TNBC patient-derived tumors (PDX), we were able to thoroughly characterize each PDX by computing a patient-specific set of unbalanced signaling processes and assigning a personalized therapy based on them. We discovered that each tumor has an average of two separate processes, and that, consistent with prior research, EGFR is a major core target in at least one of them in half of the tumors analyzed. However, anti-EGFR monotherapies were predicted to be ineffective, thus we developed personalized combination treatments based on PaSSS. These were predicted to induce anti-EGFR responses or to be used to develop an alternative therapy if EGFR was not present.In-vivo experimental validation of the predicted therapy showed that PaSSS predictions were more accurate than other therapies. Thus, we suggest that a detailed identification of molecular imbalances is necessary to tailor therapy for each TNBC. In summary, we propose a new strategy to design personalized therapy for TNBC using pY proteomics and PaSSS analysis. This method can be applied to different cancer types to improve response to the biomarker-based treatment.

Distinct breast cancer phenotypes in BRCA 1/2 carriers based on ER status.

PURPOSE: BRCA1/2 genes are the two main genes associated with hereditary breast cancers (BC). In the present study, we explore clinical and molecular characteristics of BRCA-associated BC in relation to estrogen receptor (ER) status. METHODS: Three BC databases (DB) were evaluated: (i) Hadassah oncogenetics (n = 4826); (ii) METABRIC (n = 1980), and (iii) Nick-Zainal (n = 560). We evaluated age at diagnosis in BRCA positive (BP) and BRCA negative (BN) patients, and tested for mutational signature differences in cohort iii. mRNA differential expression analysis (DEA) and pathway analysis were performed in cohort ii. RESULTS: Age at diagnosis was lower in BP vs. BN tumors in all cohorts in the ER- group, and only in cohort i for the ER + group. Signature 3 was universal in BP BC, whereas several signatures were associated with ER status. Pathway analysis was performed between BP&BN, and was significant only in ER- tumors: the major activated pathways involved cancer-related processes and were highly significant. The most significant pathway was estrogen-mediated S-phase entry and the most activated upstream regulator was ERBB2. CONCLUSION: Signature 3 was universal for all BP BC, while other signatures were associated with ER status. ER + BP& BN show similar genomic characteristics, ER- BP differed markedly from BN. This suggests that the initial carcinogenic process is universal for all BRCA carriers, but further insults lead to the development of two genomically distinct subtypes ER- and ER + . This may shed light on possible mechanisms involved in BP and carry preventive and therapeutic implications.

Metabolomic profiling of triple negative breast cancer cells suggests that valproic acid can enhance the anticancer effect of cisplatin.

Cisplatin is an effective chemotherapeutic agent for treating triple negative breast cancer (TNBC). Nevertheless, cisplatin-resistance might develop during the course of treatment, allegedly by metabolic reprograming, which might influence epigenetic regulation. We hypothesized that the histone deacetylase inhibitor (HDACi) valproic acid (VPA) can counter the cisplatin-induced metabolic changes leading to its resistance. We performed targeted metabolomic and real time PCR analyses on MDA-MB-231 TNBC cells treated with cisplatin, VPA or their combination. 22 (88%) out of the 25 metabolites most significantly modified by the treatments, were acylcarnitines (AC) and three (12%) were phosphatidylcholines (PCs). The most discernible effects were up-modulation of AC by cisplatin and, contrarily, their down-modulation by VPA, which was partial in the VPA-cisplatin combination. Furthermore, the VPA-cisplatin combination increased PCs, sphingomyelins (SM) and hexose levels, as compared to the other treatments. These changes predicted modulation of different metabolic pathways, notably fatty acid degradation, by VPA. Lastly, we also show that the VPA-cisplatin combination increased mRNA levels of the fatty acid oxidation (FAO) promoting enzymes acyl-CoA synthetase long chain family member 1 (ACSL1) and decreased mRNA levels of fatty acid synthase (FASN), which is the rate limiting enzyme of long-chain fatty acid synthesis. In conclusion, VPA supplementation altered lipid metabolism, especially fatty acid oxidation and lipid synthesis, in cisplatin-treated MDA-MB-231 TNBC cells. This metabolic reprogramming might reduce cisplatin resistance. This finding may lead to the discovery of new therapeutic targets, which might reduce side effects and counter drug tolerance in TNBC patients.

Computational quantification and characterization of independently evolving cellular subpopulations within tumors is critical to inhibit anti-cancer therapy resistance.

BACKGROUND: Drug resistance continues to be a major limiting factor across diverse anti-cancer therapies. Contributing to the complexity of this challenge is cancer plasticity, in which one cancer subtype switches to another in response to treatment, for example, triple-negative breast cancer (TNBC) to Her2-positive breast cancer. For optimal treatment outcomes, accurate tumor diagnosis and subsequent therapeutic decisions are vital. This study assessed a novel approach to characterize treatment-induced evolutionary changes of distinct tumor cell subpopulations to identify and therapeutically exploit anticancer drug resistance. METHODS: In this research, an information-theoretic single-cell quantification strategy was developed to provide a high-resolution and individualized assessment of tumor composition for a customized treatment approach. Briefly, this single-cell quantification strategy computes cell barcodes based on at least 100,000 tumor cells from each experiment and reveals a cell-specific signaling signature (CSSS) composed of a set of ongoing processes in each cell. RESULTS: Using these CSSS-based barcodes, distinct subpopulations evolving within the tumor in response to an outside influence, like anticancer treatments, were revealed and mapped. Barcodes were further applied to assign targeted drug combinations to each individual tumor to optimize tumor response to therapy. The strategy was validated using TNBC models and patient-derived tumors known to switch phenotypes in response to radiotherapy (RT). CONCLUSIONS: We show that a barcode-guided targeted drug cocktail significantly enhances tumor response to RT and prevents regrowth of once-resistant tumors. The strategy presented herein shows promise in preventing cancer treatment resistance, with significant applicability in clinical use.

"High-Risk Breast Cancer Screening in BRCA1/2 Carriers Leads to Early Detection and Improved Survival After a Breast Cancer Diagnosis".

BACKGROUND: Germline BRCA1/2 pathogenic variant (PV) carriers have high lifetime risk of developing breast cancer and therefore subjected to intense lifetime screening. However, solid data on the effectiveness of high-risk screening of the BRCA1/2 carrier population is limited. PATIENTS AND METHODS: Retrospectively, we analyzed 346 women diagnosed with breast tumors. Patients were divided according to the timing of BRCA1/2 PVrecognition, before (BRCA-preDx awareness, N = 62) or after (BRCA-postDx awareness group, N = 284) cancer diagnosis. RESULTS: Median follow-up times were 131.42 and 93.77 months in the BRCA-preDx awareness and BRCA-postDx awareness groups, respectively. In the BRCA-preDx awareness group, 78.7% of the patients had invasive tumors and 21.3% were diagnosed with pure ductal carcinoma in situ. In contrast, in the BRCA-postDx awareness group over 93% of women were diagnosed with invasive cancer and only 6.4% had in situ disease. The mode of tumor detection differed significantly between the groups: 71.9% in the BRCA-postDx awareness group and 26.2% in the BRCA-preDx awareness group were diagnosed after personally palpating a lump. Tumor size and nodal involvement were significantly more favorable in the BRCA-preDx awareness group. T stage was significantly lower in the BRCA-preDx awareness group: 54.84% at T1 and 20.96% at Tis. In the BRCA-postDx awareness group, only 37.54% were at T1 and 6.49% at Tis. The N stage was also significantly lower in the BRCA-preDx awareness group: 71% had no lymph node metastases, compared with 56.1% in the BRCA-postDx awareness group. Additionally, therapeutic procedures varied between the groups: BRCA-preDx awareness group patients underwent more breast conserving surgeries. Axillary lymph node dissection was done in 38% of women in the BRCA-postDx awareness group and in only 8.7% of the BRCA-preDx awareness group patients. Interestingly, improved survival was found among patients who underwent high-risk screening (hazard ratio=0.34). CONCLUSIONS: High-risk screening might facilitate downstaging of detected breast tumor among BRCA1/2 carrier population.

Lacosamide at therapeutic concentrations induces histone hyperacetylation in vitro.

Inhibition of histone deacetylases (HDACs) and subsequent hyperacetylation of histone proteins lead to altered gene expression associated with therapeutic drug effects, but also with teratogenicity. The only US Food and Drug Administration (FDA)-approved antiepileptic drug that has been consistently shown to induce histone hyperacetylation is valproic acid. More recently, lacosamide was reported to interfere with histone modifications, but histone hyperacetylation was not demonstrated. In the current study we evaluated the effects of lacosamide on histone acetylation in vitro. MDA-MB-231 (triple-negative breast cancer) cells and human placental BeWo cells were exposed for 16 hours to 5-20 µg/ml (20-80 µm) lacosamide. Histone acetylation was evaluated by western blot analysis. We additionally measured HDAC1 activity in the presence of lacosamide. At 5, 10, and 20 µg/ml, lacosamide enhanced histone acetylation in BeWo cells by 1.7-fold (p > 0.05), 3.4-fold (p < 0.05), and 3.0-fold (p > 0.05), respectively. Histone H3 acetylation and total histones H3 and H4 levels were not significantly modified (p > 0.05). The magnitude of change in histone acetylation in MDA-MB-231 cells was smaller (p > 0.05). In contrast to valproic acid, lacosamide did not inhibit HDAC1. Our findings suggest that the effects of lacosamide on gene expression, and the related potential antitumor activity and teratogenicity, may differ from those of valproic acid.

Safety and efficacy of human embryonic stem cell-derived astrocytes following intrathecal transplantation in SOD1G93A and NSG animal models.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by the loss of MNs in the central nervous system. As MNs die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition failure. Despite the selective MN death in ALS, there is growing evidence that malfunctional astrocytes play a crucial role in disease progression. Thus, transplantation of healthy astrocytes may compensate for the diseased astrocytes. METHODS: We developed a good manufacturing practice-grade protocol for generation of astrocytes from human embryonic stem cells (hESCs). The first stage of our protocol is derivation of astrocyte progenitor cells (APCs) from hESCs. These APCs can be expanded in large quantities and stored frozen as cell banks. Further differentiation of the APCs yields an enriched population of astrocytes with more than 90% GFAP expression (hES-AS). hES-AS were injected intrathecally into hSOD1G93A transgenic mice and rats to evaluate their therapeutic potential. The safety and biodistribution of hES-AS were evaluated in a 9-month study conducted in immunodeficient NSG mice under good laboratory practice conditions. RESULTS: In vitro, hES-AS possess the activities of functional healthy astrocytes, including glutamate uptake, promotion of axon outgrowth and protection of MNs from oxidative stress. A secretome analysis shows that these hES-AS also secrete several inhibitors of metalloproteases as well as a variety of neuroprotective factors (e.g. TIMP-1, TIMP-2, OPN, MIF and Midkine). Intrathecal injections of the hES-AS into transgenic hSOD1G93A mice and rats significantly delayed disease onset and improved motor performance compared to sham-injected animals. A safety study in immunodeficient mice showed that intrathecal transplantation of hES-AS is safe. Transplanted hES-AS attached to the meninges along the neuroaxis and survived for the entire duration of the study without formation of tumors or teratomas. Cell-injected mice gained similar body weight to the sham-injected group and did not exhibit clinical signs that could be related to the treatment. No differences from the vehicle control were observed in hematological parameters or blood chemistry. CONCLUSION: Our findings demonstrate the safety and potential therapeutic benefits of intrathecal injection of hES-AS for the treatment of ALS.

Ex-vivo organ culture as potential prioritization tool for breast cancer targeted therapy.

The growing use of genomic testing presents new treatment options but also new dilemmas. We describe here a heavily-pretreated metastatic triple negative breast cancer patient who failed to respond to conventional treatment. Genomic analyses were performed that discovered several targetable alterations (e.g. FGFR1, CDK6, INSR) and created a clinical challenge - which target to target first? Our solution to this relatively common scenario was using ex-vivo organ culture (EVOC) system to prioritize treatment directed toward the best molecular target. EVOC enabled the trial of several potent targeted agents (Everolimus, Linsitinib, Palbociclib, AZD4547) and allowed semi-quantitative measurement of tumor response. The best response was to FGFR inhibitor, AZD4547. Consequently, the most accessible FGFR inhibiting agents (Pazopanib, then Nintedanib) were administered and some response was achieved. This report provides a potential rationale for utilizing EVOC system to predict tumor response to targeted therapy when multiple targets are proposed.

Expression level of miRNAs on chromosome 14q32.31 region correlates with tumor aggressiveness and survival of glioblastoma patients.

The 54 microRNAs (miRNAs) within the DLK-DIO3 genomic region on chromosome 14q32.31 (cluster-14-miRNAs) are organized into sub-clusters 14A and 14B. These miRNAs are downregulated in glioblastomas and might have a tumor suppressive role. Any association between the expression levels of cluster-14-miRNAs with overall survival (OS) is undetermined. We randomly selected miR-433, belonging to sub-cluster 14A and miR-323a-3p and miR-369-3p, belonging to sub-cluster 14B, and assessed their role in glioblastomas in vitro and in vivo. We also determined the expression level of cluster-14-miRNAs in 27 patients with newly diagnosed glioblastoma, and analyzed the association between their level of expression and OS. Overexpression of miR-323a-3p and miR-369-3p, but not miR-433, in glioblastoma cells inhibited their proliferation and migration in vitro. Mice implanted with glioblastoma cells overexpressing miR323a-3p and miR369-3p, but not miR433, exhibited prolonged survival compared to controls (P = .003). Bioinformatics analysis identified 13 putative target genes of cluster-14-miRNAs, and real-time RT-PCR validated these findings. Pathway analysis of the putative target genes identified neuregulin as the most enriched pathway. The expression level of cluster-14-miRNAs correlated with patients' OS. The median OS was 8.5 months for patients with low expression levels and 52.7 months for patients with high expression levels (HR 0.34; 95 % CI 0.12-0.59, P = .003). The expression level of cluster-14-miRNAs correlates directly with OS, suggesting a role for this cluster in promoting aggressive behavior of glioblastoma, possibly through ErBb/neuregulin signaling.

Gliomas display a microRNA expression profile reminiscent of neural precursor cells.

Gliomas express many genes that play a role in neural precursor cells (NPCs), but no direct comparison between glioma and stem cell (SC) gene expression profiles has been performed. To investigate the similarities and differences between gliomas and SCs, we compared the microRNA (miRNA) expression signatures of glial tumors, embryonic SCs (ESCs), NPCs, and normal adult brains from both human and mouse tissues. We demonstrated that both human gliomas (regardless of their grade) and methylcholanthrene-induced mouse glioma shared an miRNA expression profile that is reminiscent of NPCs. About half of the miRNAs expressed in the shared profile clustered in seven genomic regions susceptible to genetic/epigenetic alterations in various cancers. These clusters comprised the miR17 family, mir183-182, and the SC-specific clusters mir367-302 and mir371-373, which are upregulated in gliomas, ESCs, and NPCs. The bipartite cluster of 7 + 46 miRNAs on chromosome 14q32.31, which might represent the largest tumor suppressor miRNA cluster, was downregulated in the shared expression profile. This study provides the first evidence for association between these clusters and gliomas. Despite the broad similarity in the miRNA expression profiles, 15 miRNAs showed disparate expression between SC and gliomas. Ten miRNAs belong to the 2 SC-specific clusters and the remaining (mir135b, mir141, mir205, mir200C, and mir301a) have been previously shown to associate with malignancies. Our finding showed that all gliomas displayed NPC-like miRNA signatures, which may have implications for studies of glioma origins. Furthermore, careful study of the 15 miRNAs that differ in expression between SCs and gliomas, particularly those 5 that are not SC-specific, may enhance our understanding of gliomagenesis.