Targeting neddylation and sumoylation in chemoresistant triple negative breast cancer.

Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.

BRCA1-Mediated Dual Regulation of Ferroptosis Exposes a Vulnerability to GPX4 and PARP Co-Inhibition in BRCA1-Deficient Cancers.

Resistance to poly (ADP-ribose) polymerase inhibitors (PARPi) limits the therapeutic efficacy of PARP inhibition in treating breast cancer susceptibility gene 1 (BRCA1)-deficient cancers. Here we reveal that BRCA1 has a dual role in regulating ferroptosis. BRCA1 promotes the transcription of voltage-dependent anion channel 3 (VDAC3) and glutathione peroxidase 4 (GPX4); consequently, BRCA1 deficiency promotes cellular resistance to erastin-induced ferroptosis but sensitizes cancer cells to ferroptosis induced by GPX4 inhibitors (GPX4i). In addition, nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and defective GPX4 induction unleash potent ferroptosis in BRCA1-deficient cancer cells upon PARPi and GPX4i co-treatment. Finally, we show that xenograft tumors derived from patients with BRCA1-mutant breast cancer with PARPi resistance exhibit decreased GPX4 expression and high sensitivity to PARP and GPX4 co-inhibition. Our results show that BRCA1 deficiency induces a ferroptosis vulnerability to PARP and GPX4 co-inhibition and inform a therapeutic strategy for overcoming PARPi resistance in BRCA1-deficient cancers. Significance: BRCA1 deficiency promotes resistance to erastin-induced ferroptosis via blocking VDAC3 yet renders cancer cells vulnerable to GPX4i-induced ferroptosis via inhibiting GPX4. NCOA4 induction and defective GPX4 further synergizes GPX4i with PARPi to induce ferroptosis in BRCA1-deficient cancers and targeting GPX4 mitigates PARPi resistance in those cancers. See related commentary by Alborzinia and Friedmann Angeli, p. 1372.

Predictors of success in establishing orthotopic patient-derived xenograft models of triple negative breast cancer.

Patient-derived xenograft (PDX) models of breast cancer are an effective discovery platform and tool for preclinical pharmacologic testing and biomarker identification. We established orthotopic PDX models of triple negative breast cancer (TNBC) from the primary breast tumors of patients prior to and following neoadjuvant chemotherapy (NACT) while they were enrolled in the ARTEMIS trial (NCT02276443). Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), and in cases of AC-resistance, after a 3-month course of different experimental therapies and/or additional chemotherapy (post-NACT). Our study cohort includes a total of 269 fine needle aspirates (FNAs) from 217 women, generating a total of 62 PDX models (overall success-rate = 23%). Success of PDX engraftment was generally higher from those cancers that proved to be treatment-resistant, whether poorly responsive to AC as determined by ultrasound measurements mid-NACT (p = 0.063), RCB II/III status after NACT (p = 0.046), or metastatic relapse within 2 years of surgery (p = 0.008). TNBC molecular subtype determined from gene expression microarrays of pre-NACT tumors revealed no significant association with PDX engraftment rate (p = 0.877). Finally, we developed a statistical model predictive of PDX engraftment using percent Ki67 positive cells in the patient's diagnostic biopsy, positive lymph node status at diagnosis, and low volumetric reduction of the patient's tumor following AC treatment. This novel bank of 62 PDX models of TNBC provides a valuable resource for biomarker discovery and preclinical therapeutic trials aimed at improving neoadjuvant response rates for patients with TNBC.

Identification of biomarkers of response to preoperative talazoparib monotherapy in treatment naïve gBRCA+ breast cancers.

Germline mutations in BRCA1 or BRCA2 exist in ~2-7% of breast cancer patients, which has led to the approval of PARP inhibitors in the advanced setting. We have previously reported a phase II neoadjuvant trial of single agent talazoparib for patients with germline BRCA pathogenic variants with a pathologic complete response (pCR) rate of 53%. As nearly half of the patients treated did not have pCR, better strategies are needed to overcome treatment resistance. To this end, we conducted multi-omic analysis of 13 treatment naïve breast cancer tumors from patients that went on to receive single-agent neoadjuvant talazoparib. We looked for biomarkers that were predictive of response (assessed by residual cancer burden) after 6 months of therapy. We found that all resistant tumors exhibited either the loss of SHLD2, expression of a hypoxia signature, or expression of a stem cell signature. These results indicate that the deep analysis of pre-treatment tumors can identify biomarkers that are predictive of response to talazoparib and potentially other PARP inhibitors, and provides a framework that will allow for better selection of patients for treatment, as well as a roadmap for the development of novel combination therapies to prevent emergence of resistance.

Metabolic stress induces GD2+ cancer stem cell-like phenotype in triple-negative breast cancer.

BACKGROUND: Metabolic stress resulting from nutrient deficiency is one of the hallmarks of a growing tumour. Here, we tested the hypothesis that metabolic stress induces breast cancer stem-like cell (BCSC) phenotype in triple-negative breast cancer (TNBC). METHODS: Flow cytometry for GD2 expression, mass spectrometry and Ingenuity Pathway Analysis for metabolomics, bioinformatics, in vitro tumorigenesis and in vivo models were used. RESULTS: Serum/glucose deprivation not only increased stress markers but also enhanced GD2+ BCSC phenotype and function in TNBC cells. Global metabolomics profiling identified upregulation of glutathione biosynthesis in GD2high cells, suggesting a role of glutamine in the BCSC phenotype. Cueing from the upregulation of the glutamine transporters in primary breast tumours, inhibition of glutamine uptake using small-molecule inhibitor V9302 reduced GD2+ cells by 70-80% and BCSC characteristics in TNBC cells. Mechanistic studies revealed inhibition of the mTOR pathway and induction of ferroptosis by V9302 in TNBC cells. Finally, inhibition of glutamine uptake significantly reduced in vivo tumour growth in a TNBC patient-derived xenograft model using NSG (non-obese diabetic/severe combined immunodeficiency with a complete null allele of the IL-2 receptor common gamma chain) mice. CONCLUSION: Here, we show metabolic stress results in GD2+ BCSC phenotype in TNBC and glutamine contributes to GD2+ phenotype, and targeting the glutamine transporters could complement conventional chemotherapy in TNBC.

HIF2 Regulates Intestinal Wnt5a Expression.

Radiation therapy for abdominal tumors is challenging because the small intestine is exquisitely radiosensitive. Unfortunately, there are no FDA-approved therapies to prevent or mitigate GI radiotoxicity. The EGLN protein family are oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs). Our group has previously shown that stabilization of HIF2 through genetic deletion or pharmacologic inhibition of the EGLNs mitigates and protects against GI radiotoxicity in mice by improving intestinal crypt stem cell survival. Here we aimed to elucidate the molecular mechanisms by which HIF2 confers GI radioprotection. We developed duodenal organoids from mice, transiently overexpressed non-degradable HIF2, and performed bulk RNA sequencing. Interestingly, HIF2 upregulated known radiation modulators and genes involved in GI homeostasis, including Wnt5a. Non-canonical Wnt5a signaling has been shown by other groups to improve intestinal crypt regeneration in response to injury. Here we show that HIF2 drives Wnt5a expression in multiple duodenal organoid models. Luciferase reporter assays performed in human cells showed that HIF2 directly activates the WNT5A promoter via a hypoxia response element. We then evaluated crypt regeneration using spheroid formation assays. Duodenal organoids that were pre-treated with recombinant Wnt5a had a higher cryptogenic capacity after irradiation, compared to vehicle-treated organoids. Conversely, we found that Wnt5a knockout decreased the cryptogenic potential of intestinal stem cells following irradiation. Treatment with recombinant Wnt5a prior to irradiation rescued the cryptogenic capacity of Wnt5a knockout organoids, indicating that Wnt5a is necessary and sufficient for duodenal radioprotection. Taken together, our results suggest that HIF2 radioprotects the GI tract by inducing Wnt5a expression.

The cytosolic iron-sulfur cluster assembly (CIA) pathway is required for replication stress tolerance of cancer cells to Chk1 and ATR inhibitors.

The relationship between ATR/Chk1 activity and replication stress, coupled with the development of potent and tolerable inhibitors of this pathway, has led to the clinical exploration of ATR and Chk1 inhibitors (ATRi/Chk1i) as anticancer therapies for single-agent or combinatorial application. The clinical efficacy of these therapies relies on the ability to ascertain which patient populations are most likely to benefit, so there is intense interest in identifying predictive biomarkers of response. To comprehensively evaluate the components that modulate cancer cell sensitivity to replication stress induced by Chk1i, we performed a synthetic-lethal drop-out screen in a cell line derived from a patient with triple-negative breast cancer (TNBC), using a pooled barcoded shRNA library targeting ~350 genes involved in DNA replication, DNA damage repair, and cycle progression. In addition, we sought to compare the relative requirement of these genes when DNA fidelity is challenged by clinically relevant anticancer breast cancer drugs, including cisplatin and PARP1/2 inhibitors, that have different mechanisms of action. This global comparison is critical for understanding not only which agents should be used together for combinatorial therapies in breast cancer patients, but also the genetic context in which these therapies will be most effective, and when a single-agent therapy will be sufficient to provide maximum therapeutic benefit to the patient. We identified unique potentiators of response to ATRi/Chk1i and describe a new role for components of the cytosolic iron-sulfur assembly (CIA) pathway, MMS19 and CIA2B-FAM96B, in replication stress tolerance of TNBC.

Mammary-specific expression of Trim24 establishes a mouse model of human metaplastic breast cancer.

Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.

Single-cell evaluation reveals shifts in the tumor-immune niches that shape and maintain aggressive lesions in the breast.

There is an unmet clinical need for stratification of breast lesions as indolent or aggressive to tailor treatment. Here, single-cell transcriptomics and multiparametric imaging applied to a mouse model of breast cancer reveals that the aggressive tumor niche is characterized by an expanded basal-like population, specialization of tumor subpopulations, and mixed-lineage tumor cells potentially serving as a transition state between luminal and basal phenotypes. Despite vast tumor cell-intrinsic differences, aggressive and indolent tumor cells are functionally indistinguishable once isolated from their local niche, suggesting a role for non-tumor collaborators in determining aggressiveness. Aggressive lesions harbor fewer total but more suppressed-like T cells, and elevated tumor-promoting neutrophils and IL-17 signaling, disruption of which increase tumor latency and reduce the number of aggressive lesions. Our study provides insight into tumor-immune features distinguishing indolent from aggressive lesions, identifies heterogeneous populations comprising these lesions, and supports a role for IL-17 signaling in aggressive progression.

Anti-GD2 antibody dinutuximab inhibits triple-negative breast tumor growth by targeting GD2+ breast cancer stem-like cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no effective standard therapy. Breast cancer stem-like cells (BCSCs) in primary TNBCs are reported to be responsible for metastatic spread of the disease and resistance to chemotherapy, but no available therapeutic tools target BCSCs. We previously reported that the ganglioside GD2 is highly expressed on BCSCs and that inhibition of its expression hampers TNBC growth. We therefore hypothesized that the anti-GD2 antibody dinutuximab (ch14.18) targets GD2+ BCSCs and inhibits TNBC growth. METHOD: To test our hypothesis, we first determined GD2 expression via immunohistochemistry in frozen primary tumor samples from patients with TNBC (n=89). Then, we examined the effects of dinutuximab on TNBC cell adhesion, migration, and mammosphere formation in vitro and on tumor growth in vivo using TNBC cell-line and patient-derived xenograft (PDX) models. RESULTS: We found that GD2 was expressed in around 60% of primary TNBC tumors at variable levels and was associated with worse overall survival of patients with TNBC (p=0.002). GD2 was found to be expressed in tumors and stroma, but normal ducts and lobules in adjacent tissues have shown low or no GD2 staining, indicating that GD2 is potentially a novel biomarker for tumor and its microenvironment. Treatment with dinutuximab significantly decreased adhesion and migration of MDA-MB-231 and SUM159 TNBC cells. Moreover, dinutuximab treatment inhibited mTOR signaling, which has been shown to be regulated by GD2 in BCSCs. Dinutuximab also reduced tumor growth in nude mice bearing TNBC cell-line xenografts. Finally, dinutuximab in combination with activated natural killer cells inhibited tumor growth in a TNBC PDX model and improved overall survival of tumor-bearing mice. CONCLUSIONS: Dinutuximab successfully eliminated GD2+ cells and reduced tumor growth in both in vivo models. Our data provide proof-of-concept for the criticality of GD2 in BCSCs and demonstrate the potential of dinutuximab as a novel therapeutic approach for TNBC.

A comprehensive overview of metaplastic breast cancer: clinical features and molecular aberrations.

Metaplastic breast cancer (MpBC) is an exceedingly rare breast cancer variant that is therapeutically challenging and aggressive. MpBC is defined by the histological presence of at least two cellular types, typically epithelial and mesenchymal components. This variant harbors a triple-negative breast cancer (TNBC) phenotype, yet has a worse prognosis and decreased survival compared to TNBC. There are currently no standardized treatment guidelines specifically for MpBC. However, prior studies have found that MpBC typically has molecular alterations in epithelial-to-mesenchymal transition, amplification of epidermal growth factor receptor, PI3K/Akt signaling, nitric oxide signaling, Wnt/ß-catenin signaling, altered immune response, and cell cycle dysregulation. Some of these molecular alterations have been studied as therapeutic targets, in both the preclinical and clinical setting. This current review discusses the histological organization and cellular origins of MpBC, molecular alterations, the role of radiation therapy, and current clinical trials for MpBC.

Pharmacologic profiling of patient-derived xenograft models of primary treatment-naïve triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States, lacks targeted therapeutic options, and is associated with a 40-80% risk of recurrence. Thus, identifying actionable targets in treatment-naïve and chemoresistant TNBC is a critical unmet medical need. To address this need, we performed high-throughput drug viability screens on human tumor cells isolated from 16 patient-derived xenograft models of treatment-naïve primary TNBC. The models span a range of TNBC subtypes and exhibit a diverse set of putative driver mutations, thus providing a unique patient-derived, molecularly annotated pharmacologic resource that is reflective of TNBC. We identified therapeutically actionable targets including kinesin spindle protein (KSP). The KSP inhibitor targets the mitotic spindle through mechanisms independent of microtubule stability and showed efficacy in models that were resistant to microtubule inhibitors used as part of the current standard of care for TNBC. We also observed subtype selectivity of Prima-1Met, which showed higher levels of efficacy in the mesenchymal subtype. Coupling pharmacologic data with genomic and transcriptomic information, we showed that Prima-1Met activity was independent of its canonical target, mutant p53, and was better associated with glutathione metabolism, providing an alternate molecularly defined biomarker for this drug.

The RNA-binding protein AKAP8 suppresses tumor metastasis by antagonizing EMT-associated alternative splicing.

Alternative splicing has been shown to causally contribute to the epithelial-mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein-protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.

Neoadjuvant Talazoparib for Patients With Operable Breast Cancer With a Germline BRCA Pathogenic Variant.

PURPOSE: Talazoparib has demonstrated efficacy in patients with BRCA-positive metastatic breast cancer. This study evaluated the pathologic response of talazoparib alone for 6 months in patients with a known germline BRCA pathogenic variant (gBRCA-positive) and operable breast cancer. METHODS: Eligibility included 1 cm or larger invasive tumor and gBRCA-positive disease. Human epidermal growth factor receptor 2-positive tumors were excluded. Twenty patients underwent a pretreatment biopsy, 6 months of once per day oral talazoparib (1 mg), followed by definitive surgery. Patients received adjuvant therapy at physician's discretion. The primary end point was residual cancer burden (RCB). With 20 patients, the RCB-0 plus RCB-I response rate can be estimated with a 95% CI with half width less than 20%. RESULTS: Twenty patients were enrolled from August 2016 to September 2017. Median age was 38 years (range, 23 to 58 years); 16 patients were gBRCA1 positive and 4 patients were gBRCA2 positive. Fifteen patients had triple-negative breast cancer (estrogen receptor/progesterone receptor < 10%), and five had hormone receptor-positive disease. Five patients had clinical stage I disease, 12 had stage II, and three had stage III, including one patient with inflammatory breast carcinoma and one with metaplastic chondrosarcomatous carcinoma. One patient chose to receive chemotherapy before surgery and was not included in RCB analyses. RCB-0 (pathologic complete response) rate was 53% and RCB-0/I was 63%. Eight patients (40%) had grade 3 anemia and required a transfusion, three patients had grade 3 neutropenia, and 1 patient had grade 4 thrombocytopenia. Common grade 1 or 2 toxicities were nausea, fatigue, neutropenia, alopecia, dizziness, and dyspnea. Toxicities were managed by dose reduction and transfusions. Nine patients required dose reduction. CONCLUSION: Neoadjuvant single-agent oral talazoparib once per day for 6 months without chemotherapy produced substantial RCB-0 rate with manageable toxicity. The substantive pathologic response to single-agent talazoparib supports the larger, ongoing neoadjuvant trial (ClinicalTrials.gov identifier: NCT03499353).

6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase-2 Regulates TP53-Dependent Paclitaxel Sensitivity in Ovarian and Breast Cancers.

PURPOSE: Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases.Experimental Design: Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of PFKFB2 on tumor cell growth in vivo. RESULTS: Knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP53). Silencing PFKFB2 significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of siPFKFB2 increased the glycolysis rate, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cells with wtTP53, decreasing NADPH. ROS accumulated after PFKFB2 knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma. CONCLUSIONS: PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wtTP53.

Fasting Reduces Intestinal Radiotoxicity, Enabling Dose-Escalated Radiation Therapy for Pancreatic Cancer.

PURPOSE: Chemotherapy combined with radiation therapy is the most commonly used approach for treating locally advanced pancreatic cancer. The use of curative doses of radiation in this disease setting is constrained because of the close proximity of the head of the pancreas to the duodenum. The purpose of this study was to determine whether fasting protects the duodenum from high-dose radiation, thereby enabling dose escalation for efficient killing of pancreatic tumor cells. METHODS AND MATERIALS: C57BL/6J mice were either fed or fasted for 24 hours and then exposed to total abdominal radiation at 11.5 Gy. Food intake, body weight, overall health, and survival were monitored. Small intestines were harvested at various time points after radiation, and villi length, crypt depth, and number of crypts per millimeter of intestine were determined. Immunohistochemistry was performed to assess apoptosis and double-strand DNA breaks, and microcolony assays were performed to determine intestinal stem cell regeneration capacity. A syngeneic KPC model of pancreatic cancer was used to determine the effects of fasting on the radiation responses of both pancreatic cancer and host intestinal tissues. RESULTS: We demonstrated that a 24-hour fast in mice improved intestinal stem cell regeneration, as revealed by microcolony assay, and improved host survival of lethal doses of total abdominal irradiation compared with fed controls. Fasting also improved survival of mice with orthotopic pancreatic tumors subjected to lethal abdominal radiation compared with controls with free access to food. Furthermore, fasting did not affect tumor cell killing by radiation therapy and enhanced γ-H2AX staining after radiation therapy, suggesting an additional mild radiosensitizing effect. CONCLUSIONS: These results establish proof of concept for fasting as a dose-escalation strategy, enabling ablative radiation in the treatment of unresectable pancreatic cancer.

Sphingosine Kinase 1 Signaling Promotes Metastasis of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. To identify TNBC therapeutic targets, we performed integrative bioinformatics analysis of multiple breast cancer patient-derived gene expression datasets and focused on kinases with FDA-approved or in-pipeline inhibitors. Sphingosine kinase 1 (SPHK1) was identified as a top candidate. SPHK1 overexpression or downregulation in human TNBC cell lines increased or decreased spontaneous metastasis to lungs in nude mice, respectively. SPHK1 promoted metastasis by transcriptionally upregulating the expression of the metastasis-promoting gene FSCN1 via NFκB activation. Activation of the SPHK1/NFκB/FSCN1 signaling pathway was associated with distance metastasis and poor clinical outcome in patients with TNBC. Targeting SPHK1 and NFκB using clinically applicable inhibitors (safingol and bortezomib, respectively) significantly inhibited aggressive mammary tumor growth and spontaneous lung metastasis in orthotopic syngeneic TNBC mouse models. These findings highlight SPHK1 and its downstream target, NFκB, as promising therapeutic targets in TNBC. SIGNIFICANCE: SPHK1 is overexpressed in TNBC and promotes metastasis, targeting SPHK1 or its downstream target NFκB with clinically available inhibitors could be effective for inhibiting TNBC metastasis.

Ca2+-Stimulated AMPK-Dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection.

Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.

Selective EGLN Inhibition Enables Ablative Radiotherapy and Improves Survival in Unresectable Pancreatic Cancer.

When pancreatic cancer cannot be removed surgically, patients frequently experience morbidity and death from progression of their primary tumor. Radiation therapy (RT) cannot yet substitute for an operation because radiation causes fatal bleeding and ulceration of the nearby stomach and intestines before achieving tumor control. There are no FDA-approved medications that prevent or reduce radiation-induced gastrointestinal injury. Here, we overcome this fundamental problem of anatomy and biology with the use of the oral EGLN inhibitor FG-4592, which selectively protects the intestinal tract from radiation toxicity without protecting tumors. A total of 70 KPC mice with autochthonous pancreatic tumors received oral FG-4592 or vehicle control ± ablative RT to a cumulative 75 Gy administered in 15 daily fractions to a limited tumor field. Although ablative RT reduced complications from local tumor progression, fatal gastrointestinal bleeding was observed in 56% of mice that received high-dose RT with vehicle control. However, radiation-induced bleeding was completely ameliorated in mice that received high-dose RT with FG-4592 (0% bleeding, P < 0.0001 compared with vehicle). Furthermore, FG-4592 reduced epithelial apoptosis by half (P = 0.002) and increased intestinal microvessel density by 80% compared with vehicle controls. EGLN inhibition did not stimulate cancer growth, as treatment with FG-4592 alone, or overexpression of HIF2 within KPC tumors independently improved survival. Thus, we provide a proof of concept for the selective protection of the intestinal tract by the EGLN inhibition to enable ablative doses of cytotoxic therapy in unresectable pancreatic cancer by reducing untoward morbidity and death from radiation-induced gastrointestinal bleeding. SIGNIFICANCE: Selective protection of the intestinal tract by EGLN inhibition enables potentially definitive doses of radiation therapy. This might allow radiation to be a surgical surrogate for unresectable pancreatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2327/F1.large.jpg.