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Deregulated alternative splicing of the endocytic adaptor NUMB resulting in high expression of Exon9in (exon 9-containing) isoforms has been reported in several cancer types. However, the role of Numb isoform expression in tumor progression and the underlying mechanisms remain elusive. Here, we report greater exon 9 inclusion in multiple cancer types including all subtypes of breast cancer, and correlation of higher exon 9 inclusion in patients with worse prognosis. Deletion of Exon9in in breast cancer cells leads to reduced cell growth and a significant decrease of lung metastasis in orthotopic xenograft experiments. Quantitative mass spectrometry revealed downregulation of proteins involved in EMT and ECM organization and remodeling of the endocytic protein network in cells lacking the Exon9in Numb isoforms. Exon 9 deletion also results in reduced surface levels of ITGβ5, and downstream signaling to ERK and SRC, consistent with enhance lysosomal targeting mediated by the remaining Exon9sk (exon 9 skipping) Numb isoforms.SIGNIFICANCEExpression of NUMB Exon9in protein isoforms correlate with worse progression free survival, particularly in breast cancer. Our findings also reveal that Exon9in isoforms promote breast cancer progression by relieving Numb mediated down regulation of integrins and implicate Numb alternative splicing as a progression factor in multiple cancer types.

Small molecule inhibitors of BRAF and MEK have proven effective at inhibiting tumor growth in melanoma patients, however this efficacy is limited due to the almost universal development of drug resistance. To provide advanced insight into the signaling responses that occur following kinase inhibition we have performed quantitative (phospho)-proteomics of human melanoma cells treated with either dabrafenib, a BRAF inhibitor; trametinib, a MEK inhibitor or SCH772984, an ERK inhibitor. Over nine experiments we identified 7827 class I phosphorylation sites on 4960 proteins. This included 54 phosphorylation sites that were significantly down-modulated after exposure to all three inhibitors, 34 of which have not been previously reported. Functional analysis of these novel ERK targets identified roles for them in GTPase activity and regulation, apoptosis and cell-cell adhesion. Comparison of the results presented here with previously reported phosphorylation sites downstream of ERK showed a limited degree of overlap suggesting that ERK signaling responses may be highly cell line and cue specific. In addition we identified 26 phosphorylation sites that were only responsive to dabrafenib. We provide further orthogonal experimental evidence for 3 of these sites in human embryonic kidney cells over-expressing BRAF as well as further computational insights using KinomeXplorer. The validated phosphorylation sites were found to be involved in actin regulation, which has been proposed as a novel mechanism for inhibiting resistance development. These results would suggest that the linearity of the BRAF-MEK-ERK module is at least context dependent.

On-patent and off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we conducted chemical screens and identified the antihelmintic flubendazole. Flubendazole induced cell death in leukemia and myeloma cell lines and primary patient samples at nanomolar concentrations. Moreover, it delayed tumor growth in leukemia and myeloma xenografts without evidence of toxicity. Mechanistically, flubendazole inhibited tubulin polymerization by binding tubulin at a site distinct from vinblastine. In addition, cells resistant to vinblastine because of overexpression of P-glycoprotein remained fully sensitive to flubendazole, indicating that flubendazole can overcome some forms of vinblastine resistance. Given the different mechanisms of action, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. Flubendazole synergized with vinblastine to reduce the viability of OCI-AML2 cells. In addition, combinations of flubendazole with vinblastine or vincristine in a leukemia xenograft model delayed tumor growth more than either drug alone. Therefore, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines with little or no effect on normal cells. However, its effect is limited as some cancers including pancreatic cancer show de novo resistance to TRAIL induced apoptosis. In this study we report that GSK-3 inhibition using the pharmacologic agent AR-18, enhanced TRAIL sensitivity in a range of pancreatic and prostate cancer cell lines. This sensitization was found to be caspase-dependent, and both pharmacological and genetic knock-down of GSK-3 isoforms resulted in apoptotic features as shown by cleavage of PARP and caspase-3. Elevated levels of reactive oxygen intermediates and disturbance of mitochondrial membrane potential point to a mitochondrial amplification loop for TRAIL-induced apoptosis after GSK-3 inhibition. Consistent with this, overexpression of anti-apoptotic mitochondrial targets such as Bcl-XL, Mcl-1, and Bcl-2 rescued PANC-1 and PPC-1 cells from TRAIL sensitization. However, overexpression of the caspase-8 inhibitor CrmA also inhibited the sensitizing effects of GSK-3 inhibitor, suggesting an additional role for GSK-3 that inhibits death receptor signaling. Acute treatment of mice bearing PANC-1 xenografts with a combination of AR-18 and TRAIL also resulted in a significant increase in apoptosis, as measured by caspase-3 cleavage. Sensitization to TRAIL occurred despite an increase in β-catenin due to GSK-3 inhibition, suggesting that the approach might be effective even in cancers with dysregulated β-catenin. These results suggest that GSK-3 inhibitors might be effectively combined with TRAIL for the treatment of pancreatic cancer.

Normal epithelial cells undergo apoptosis upon detachment from the extracellular matrix, a process termed “anoikis.” However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. Molecules that sensitize resistant cells to anoikis will be useful chemical probes to understand this pathway. To identify novel anoikis sensitizers in anoikis-resistant PPC-1 prostate adenocarcinoma cells, a library of 2,000 off-patent drugs and natural products was screened for their ability to preferentially induce cell death in suspension over adherent culture conditions. This screen identified five members of the family of cardiac glycosides as anoikis sensitizers, including ouabain, peruvoside, digoxin, digitoxin, and strophanthidin. We conducted further studies with ouabain to discern the mechanism of cardiac glycoside-induced anoikis sensitization. Ouabain initiated anoikis through the mitochondrial pathway of caspase activation. In addition, ouabain sensitized cells to anoikis by inhibiting its known target, the Na+/K+ ATPase pump, and inducing hypoosmotic stress. Resistance to anoikis permits cancer cells to survive in the circulation and facilitates their metastasis to distant organs, so we tested the effects of Na+/K+ ATPase inhibition on distant tumor formation in mouse models. In these mouse models, ouabain inhibited tumor metastases but did not alter the growth of subcutaneous tumors. Thus, we have identified a novel mechanism to sensitize resistant cells to anoikis and decrease tumor metastasis. These results suggest a potential mechanism for the observed clinical reduction in metastasis and relapse in breast cancer patients who have undergone treatments with cardiac glycosides. [Cancer Res 2009;69(7):2739–47]

To identify known drugs with unrecognized anti-leukemia activity, we compiled a library of 500 on patent and off patent compounds, and screened it to identify compounds cytotoxic to leukemia cell lines. From this screen we identified Tigecycline, an antibiotic approved for the treatment of cutaneous and intra-abdominal infections. Tigecycline induced cell death in leukemia cell lines (LD50 3 to 8 μM, n = 6 cell lines) and primary Acute Myeloid Leukemia (AML) patient samples (LD50 5-10 μM, n = 7), preferentially over normal hematopoietic cells (10% cell death at 20 μM, n = 4) by MTS assays and Annexin V staining. Likewise, in colony formation assays, Tigecycline (5μM) reduced the clonogenic growth of primary AML patient samples (n = 4) by 95±1.5 %, demonstrating an effect on leukemia progenitor cells. In contrast, 5 μM of Tigecycline reduced the clonogenic growth of normal hematopoetic cells by 34± 5% (n = 5). Although Tigecycline is structurally related to tetracycline and minocycline, these drugs were not cytotoxic towards AML cell lines up to 25 μM. Thus, Tigecycline appears preferentially cytotoxic to leukemia cells at pharmacologically achievable concentrations. Given the anti-leukemic effects of Tigecycline in vitro, we evaluated the efficacy of Tigecycline as a potential anti-leukemic agent in vivo. Mice injected subcutaneously with OCI-AML2 leukemia cells were treated with Tigecycline 50 mg/kg i.p. daily. Compared to control, Tigecycline decreased tumour mass and volume without toxicity. We also assessed the effect of Tigecycline on primary AML stem cells defined by their ability to initiate leukemic engraftment in vivo. NOD-SCID mice were injected intra-femorally with primary AML cells. Two weeks after injection, mice were treated with Tigecycline 50 mg/kg i.p. daily for two weeks. After treatment, engraftment of human AML cells was measured by flow cytometry. Compared to control, Tigecycline decreased engraftment of AML cells without toxicity. Tigecycline binds and inhibits the bacterial 30S ribosome. Bacterial ribosomes are more homologous to mitochondrial ribosomes than cytosolic ribosomes, so we compared the effects of Tigecycline on mitochondrial and cytosolic protein synthesis. At times preceding the onset of cell death, Tigecycline decreased levels of the mitochondrial protein Cox-1. In contrast, it did not decrease the expression of cytosolic short half-life proteins Bcl-XL and XIAP, suggesting a preferential effect on mitochondrial protein synthesis. Thus, Tigecycline demonstrated preclinical activity through a mechanism related to mitochondrial protein synthesis inhibition. Moreover, Tigecycline appeared cytotoxic to leukemia stem cells over normal hematopoetic stem cells. Given its prior pharmacology and toxicology testing, Tigecycline could be rapidly repositioned for a new anti-leukemic indication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4538.

Summary Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks., Graphical Abstract, Highlights • Mutations perturbing signaling networks are systematically classified and interpreted • Several such functional mutations are identified in cancer and experimentally validated • The results suggest that a single point mutant can have profound signaling effects • Systematic interpretation of genomic data may assist future precision-medicine efforts, A systematic classification of genomic variants in cancer reveals the many ways in which signaling networks can be perturbed, including rewiring and the creation or destruction of phosphorylation sites.