Engaging girls with disabilities through cellphilming: Reflections on participatory visual research as a means of countering violence in the Global South.

Despite the fact that most countries have ratified the United Nations' Convention on the Rights of the Child, girls with disabilities experience multiple forms of violence and oppression. Using a critical approach to arts-based research, we argue that while disabled girls experience multiple vulnerabilities due to the structural conditions which exposed them to violence, the use of participatory visual methods allowed them to reframe their stories. This article discusses the implications for reconceptualizing diverse childhoods in relation to methodologies for empowering disabled girls.

Specific, reversible G1 arrest by UCN-01 in vivo provides cytostatic protection of normal cells against cytotoxic chemotherapy in breast cancer.

BACKGROUND: Low-dose UCN-01 mediates G1 arrest in normal proliferating cell lines with an intact G1 to S transition but not tumour cells with a deregulated G1 to S checkpoint. Here we hypothesised that UCN-01 is effective in mediating a selective, reversible G1 arrest of normal proliferating cells, resulting in decreased chemotoxicity, improved tolerance and enhanced chemotherapeutic efficacy in vivo in both non-tumour-bearing mice and in breast cancer cell line xenograft models. METHODS: Murine small bowel epithelium was used to examine the kinetics and mechanism of low-dose UCN-01-mediated arrest of normal proliferating cells and if it can protect tumour-bearing mice (MDA-MB-468 xenografts) against the toxic effects of chemotherapy (5-fluorouricil (5-FU)) allowing for its full therapeutic activity. RESULTS: UCN-01 causes significant, reversible arrest of normal gut epithelial cells at 24 h; this arrest persists for up to 7 days. Normal cellular proliferation returns by 2 weeks. Pre-treatment of both non-tumour-bearing and MDA-MB-468 tumour-bearing mice with UCN-01 prior to bolus 5-FU (450 mg/kg) yielded enhanced therapeutic efficacy with significantly decreased tumour volumes and increased survival. CONCLUSIONS: UCN-01 mediates a specific, reversible G1 arrest of normal cells in vivo and provides a cytoprotective strategy that decreases toxicity of cytotoxic chemotherapy without compromising efficacy.

Cytoplasmic Cyclin E and Phospho-Cyclin-Dependent Kinase 2 Are Biomarkers of Aggressive Breast Cancer.

Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer.

Specific ion effects on the electrophoretic mobility of small, highly charged peptides: a modeling study.

In this work, we use coarse-grained modeling to study the free solution electrophoretic mobility of small highly charged peptides (lysine, arginine, and short oligos thereof (up to nonapeptides)) in NaCl and Na2SO4 aqueous solutions at neutral pH and room temperature. The experimental data are taken from the literature. A bead modeling methodology that treats the electrostatics at the level of the nonlinear Poisson Boltzmann equation developed previously in our laboratory is able to account for the mobility of all peptides in NaCl, but not Na2SO4. The peptide mobilities in Na2SO4 can be accounted for by including sulfate binding in the model and this is proposed as one possible explanation for the discrepancy. Oligo arginine peptides bind more sulfate than oligo lysines and sulfate binding increases with the oligo length.

Fulbright as Opportunity for U.S. Nursing Educators and International Nursing Schools.

BACKGROUND: Nursing education across the globe is rapidly evolving in terms of curricular expectations and professional preparation. While there is a plethora of curricular resources and graduate programs in the United States, in some countries, these resources are limited. METHODS: The Fulbright Specialist program, the application process, and challenges as well as the benefits of the role are described. The deliverables by the Fulbright Specialist, e.g. demonstrating classroom pedagogical methods, providing access to an online doctoral program, and explaining publication strategies, are noted. RESULTS: Immediate and 2-month follow-up information regarding the Specialist's deliverables are described. The benefits to the Specialist are also detailed. CONCLUSION: Nursing educators in the U.S. and leaders of nursing schools outside of the U.S. are invited to share pedagogical practices and provide faculty development through the Fulbright Specialist program. The benefits of a collaboration are mutually beneficial. [J Nurs Educ. 2024;63(X):XXX-XXX.].

Staurosporine is chemoprotective by inducing G1 arrest in a Chk1- and pRb-dependent manner.

Chemotherapeutic agents have been the mainstay of cancer therapy for years. However, their effectiveness has been limited by toxicities they impart on normal cells. Staurosporine (ST) has been shown to arrest normal, but not breast cancer, cells in G1. Therefore, ST may become a chemoprotective agent, arresting normal cells while allowing tumor cells to enter cell cycle phases where they are sensitive to chemotherapeutic agents. Understanding the mechanism of ST-mediated G1 arrest may allow for a beneficial chemoprotective treatment strategy for patients. We utilized 76NE6 (pRb+/p53-), 76NF2V (pRb+/p53+) and 76NE7 (pRb-/P53+) non-tumorigenic human mammary epithelial cell lines to understand the role of the Rb and p53 pathways in ST-directed G1 arrest. CDK4 was downregulated by ST in Rb+ cells, but its presence could not reverse the arrest, neither did its stable downregulation alter ST-mediated cellular response. ST-mediated G1 arrest required pRb, which in turn initiated a cascade of events leading to inhibition of CDK4. Further assessment of this pathway revealed that Chk1 expression and activity were required for the Rb-dependent arrest. For example, pRb+ cells with small interfering RNA to Chk1 had approximately 60% less cells in G1 phase compared with controls and pRb- cells do not arrest upon ST. Furthermore, Chk1 expression facilitates the release of the Rb+ cells from G1 arrest. Collectively, our data suggest that pRb cooperates with Chk1 to mediate a G1 arrest only in pRb+ cells. The elucidation of this pathway can help identify novel agents to protect cancer patients against the debilitating effects of chemotherapy.

Sequential Targeting of Retinoblastoma and DNA Synthesis Pathways Is a Therapeutic Strategy for Sarcomas That Can Be Monitored in Real Time.

Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell-cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1,043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective two-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1 phase. Upon drug removal, cells synchronously traversed to S phase, where a second treatment with S-phase targeted agents (gemcitabine or Wee1 kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be noninvasively monitored using real-time positron emission tomography imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA-damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with noninvasive functional imaging and serum biomarkers as indicators of response and cell cycling. SIGNIFICANCE: An innovative sequential therapeutic strategy targeting Rb, followed by treatment with agents that perturb DNA synthesis pathways, results in synergistic killing of Rb-positive sarcomas that can be noninvasively monitored.

Abemaciclib Is Effective in Palbociclib-Resistant Hormone Receptor-Positive Metastatic Breast Cancers.

Cyclin-dependent kinases 4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) is standard of care for patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (MBC). However, resistance to CDK4/6is plus ET remains a clinical problem with limited therapeutic options following disease progression. Different CDK4/6is might have distinct mechanisms of resistance, and therefore using them sequentially or targeting their differentially altered pathways could delay disease progression. To understand pathways leading to resistance to the CDK4/6is palbociclib and abemaciclib, we generated multiple in vitro models of palbociclib-resistant (PR) and abemaciclib-resistant (AR) cell lines as well as in vivo patient-derived xenografts (PDX) and ex vivo PDX-derived organoids (PDxO) from patients who progressed on CDK4/6i. PR and AR breast cancer cells exhibited distinct transcriptomic and proteomic profiles that sensitized them to different classes of inhibitors; PR cells upregulated G2-M pathways and responded to abemaciclib, while AR cells upregulated mediators of the oxidative phosphorylation pathway (OXPHOS) and responded to OXPHOS inhibitors. PDX and organoid models derived from patients with PR breast cancer remained responsive to abemaciclib. Resistance to palbociclib while maintaining sensitivity to abemaciclib was associated with pathway-specific transcriptional activity but was not associated with any individual genetic alterations. Finally, data from a cohort of 52 patients indicated that patients with HR-positive/HER2-negative MBC who progressed on palbociclib-containing regimens can exhibit a meaningful overall clinical benefit from abemaciclib-based therapy when administered after palbociclib. These findings provide the rationale for clinical trials evaluating the benefit of abemaciclib treatment following progression on a prior CDK4/6i. SIGNIFICANCE: Palbociclib-resistant breast cancers respond to abemaciclib and express pathway-specific signatures of sensitivity, providing a biomarker-driven therapeutic option for patients with metastatic breast cancer following disease progression on cyclin-dependent kinases 4/6 inhibitors.

Low-molecular-weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast cancer.

Low-molecular-weight cyclin E (LMW-E) is an N-terminus deleted (40 amino acid) form of cyclin E detected in breast cancer, but not in normal cells or tissues. LMW-E overexpression predicts poor survival in breast cancer patients independent of tumor proliferation rate, but the oncogenic mechanism of LMW-E and its unique function(s) independent of full-length cyclin E (FL-cycE) remain unclear. In the current study, we found LMW-E was associated with genomic instability in early-stage breast tumors (n = 725) and promoted genomic instability in human mammary epithelial cells (hMECs). Mechanistically, FL-cycE overexpression inhibited the proliferation of hMECs by replication stress and DNA damage accumulation, but LMW-E facilitated replication stress tolerance by upregulating DNA replication and damage repair. Specifically, LMW-E interacted with chromatin and upregulated the loading of minichromosome maintenance complex proteins (MCMs) in a CDC6 dependent manner and promoted DNA repair in a RAD51- and C17orf53-dependent manner. Targeting the ATR-CHK1-RAD51 pathway with ATR inhibitor (ceralasertib), CHK1 inhibitor (rabusertib), or RAD51 inhibitor (B02) significantly decreased the viability of LMW-E-overexpressing hMECs and breast cancer cells. Collectively, our findings delineate a novel role for LMW-E in tumorigenesis mediated by replication stress tolerance and genomic instability, providing novel therapeutic strategies for LMW-E-overexpressing breast cancers.

Semi-high throughput method of measuring proteasome inhibition in vitro and in cultured cells.

The ubiquitin proteasome-proteolytic pathway has emerged as one of the most significant pathways in modulating protein homeostasis under both normal and disease states. The use of proteasome inhibitors (PI) has played a pivotal role in understanding protein turn over. The main objective of this work was to develop a comprehensive, fast, and reliable, yet simple in vitro assay that would allow for the identification and characterization of a wide range of PIs. The assays consist of a 96-well plate high throughput (HTP) method to assess proteasome activity in Hs578T breast cancer cell extracts, purified 20S proteasome, using a fluorogenic substrate, Suc-leu-leu-val-tyr-7-AMC, specific to the chymotrypsin-like enzymatic activity of the proteasome. We showed that the chymotrypsin-like activity of the proteasome was inhibited in the two in vitro systems, albeit to different degrees. The assay system also includes two cell-based assays consisting of a vector expressing a fusion protein of green fluorescent protein (gfp) and Mouse Ornithine Decarboxylase (MODC) in Zs578T (parental Hs578T carrying the vector that expresses the fusion protein). In the cell-based assay analyses (qualitatively by microscopy and quantitatively by flow cytometry), treatment of Zs578T with PIs prevented the degradation of MODC, accumulated gfp, indicative of increased proteasome inhibition. Because no single assay represents a definitive proof of proteasome inhibitory activity, combined, these assays should serve as a comprehensive benchmark for the identification and partial characterization of novel inhibitors. In summary, the four-step assay protocol can easily be adapted into a high throughput format to rapidly screen unknown inhibitors.

Comparative analysis of genetic diversity of sesame (Sesamum indicum L.) from Vietnam and Cambodia using agro-morphological and molecular markers.

The purpose of this study was to comparatively analyze the genetic diversity of sesame (Sesamum indicum L.) using agro-morphological and molecular markers. Twelve sesame populations collected from three regions in Cambodia and Vietnam were used in this study. A high genetic variation was revealed both by agro-morphological and RAPD markers within and among the 12 sesame populations. The range of agro-morphological trait based average taxonomic distance among populations (0.02 to 0.47) was wider than that of RAPD based genetic distance (0.06 to 0.27). The mean distance revealed by agro-morphological markers (0.23) and RAPD markers (0.22) was similar. RAPD based analysis revealed a relatively higher genetic diversity in populations from South Vietnam as compared to the other two regions. Interestingly, populations from this region also had higher values for yield related traits such as number of capsules per plant, number of seeds per capsule, and seed yield per plant suggesting positive correlation between the extent of genetic variation within population and yield related traits in sesame. A highly significant positive correlation (r = 0.88, P < 0.001) was found between agro-morphological and RAPD markers in estimating the genetic distance between populations. Both methods suggested the existence of a substantial amount of genetic diversity both in the Vietnamese and Cambodian populations. Although both agro-morphological and RAPD markers were found to be useful in genetic diversity analysis in sesame, their combined use would give superior results.

LMW cyclin E and its novel catalytic partner CDK5 are therapeutic targets and prognostic biomarkers in salivary gland cancers.

Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.

Targeting Replicative Stress and DNA Repair by Combining PARP and Wee1 Kinase Inhibitors Is Synergistic in Triple Negative Breast Cancers with Cyclin E or BRCA1 Alteration.

The identification of biomarker-driven targeted therapies for patients with triple negative breast cancer (TNBC) remains a major clinical challenge, due to a lack of specific targets. Here, we show that cyclin E, a major regulator of G1 to S transition, is deregulated in TNBC and is associated with mutations in DNA repair genes (e.g., BRCA1/2). Breast cancers with high levels of cyclin E not only have a higher prevalence of BRCA1/2 mutations, but also are associated with the worst outcomes. Using several in vitro and in vivo model systems, we show that TNBCs that harbor either mutations in BRCA1/2 or overexpression of cyclin E are very sensitive to the growth inhibitory effects of AZD-1775 (Wee 1 kinase inhibitor) when used in combination with MK-4837 (PARP inhibitor). Combination treatment of TNBC cell lines with these two agents results in synergistic cell killing due to induction of replicative stress, downregulation of DNA repair and cytokinesis failure that results in increased apoptosis. These findings highlight the potential clinical application of using cyclin E and BRCA mutations as biomarkers to select only those patients with the highest replicative stress properties that may benefit from combination treatment with Wee 1 kinase and PARP inhibitors.

Overexpression of the low molecular weight cyclin E in transgenic mice induces metastatic mammary carcinomas through the disruption of the ARF-p53 pathway.

In tumor cells, cyclin E deregulation results in the appearance of five low molecular weight (LMW) isoforms. When overexpressed in breast cancer cells, these forms of cyclin E induce genomic instability, resistance to inhibition by p21 and p27, and resistance to antiestrogen therapy. Additionally, the LMW forms of cyclin E strongly correlate with decreased survival in patients with breast cancer. However, the oncologic role of the LMW forms of cyclin E in breast cancer tumorigenesis is yet to be determined. To this end, we generated transgenic mice expressing full-length cyclin E alone (M46A), full-length and the EL4 isoforms (EL1/EL4), or the EL2/3 isoforms of cyclin E (T1) under the control of the mouse mammary tumor virus promoter. Compared with full-length cyclin E, LMW cyclin E overexpression induces delayed mammary growth during the pubertal phase and abnormal cell morphology during lactation. Both primary mammary tumor formation and metastasis were markedly enhanced in LMW cyclin E transgenic mice. LMW cyclin E overexpression in mammary epithelial cells of mice is sufficient by itself to induce mammary adenocarcinomas in 34 of 124 (27%) animals compared with 7 of 67 (10.4%) mice expressing only the full-length cyclin E (P < 0.05). In addition, metastasis was seen in 25% of LMW cyclin E tumor-bearing animals compared with only 8.3% of tumors in the full-length cyclin E background (P < 0.05). Moreover, LMW cyclin E overexpression selects for inactivation of p53 by loss of heterozygosity and spontaneous and frequent inactivation of ARF. Therefore, LMW cyclin E overexpression strongly selects for spontaneous inactivation of the ARF-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy.

Combined Inhibition of STAT3 and DNA Repair in Palbociclib-Resistant ER-Positive Breast Cancer.

PURPOSE: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are currently used in combination with endocrine therapy to treat advanced hormone receptor-positive, HER2-negative breast cancer. Although this treatment doubles time to progression compared with endocrine therapy alone, about 25%-35% of patients do not respond, and almost all patients eventually acquire resistance. Discerning the mechanisms of resistance to CDK4/6 inhibition is crucial in devising alternative treatment strategies. EXPERIMENTAL DESIGN: Palbociclib-resistant cells (MCF-7 and T47D) were generated in a step-wise dose-escalading fashion. Whole-exome sequencing, genome-wide expression analysis, and proteomic analysis were performed in both resistant and parental (sensitive) cells. Pathway alteration was assessed mechanistically and pharmacologically. Biomarkers of altered pathways were examined in tumor samples from patients with palbociclib-treated breast cancer whose disease progressed while on treatment. RESULTS: Palbociclib-resistant cells are cross-resistant to other CDK4/6 inhibitors and are also resistant to endocrine therapy (estrogen receptor downregulation). IL6/STAT3 pathway is induced, whereas DNA repair and estrogen receptor pathways are downregulated in the resistant cells. Combined inhibition of STAT3 and PARP significantly increased cell death in the resistant cells. Matched tumor samples from patients with breast cancer who progressed on palbociclib were examined for deregulation of estrogen receptor, DNA repair, and IL6/STAT3 signaling, and results revealed that these pathways are all altered as compared with the pretreatment tumor samples. CONCLUSIONS: Palbociclib resistance induces endocrine resistance, estrogen receptor downregulation, and alteration of IL6/STAT3 and DNA damage response pathways in cell lines and patient samples. Targeting IL6/STAT3 activity and DNA repair deficiency using a specific STAT3 inhibitor combined with a PARP inhibitor could effectively treat acquired resistance to palbociclib.

Synthetic Lethality of PARP Inhibitors in Combination with MYC Blockade Is Independent of BRCA Status in Triple-Negative Breast Cancer.

PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients. Several of those patients exhibit intrinsic/acquired resistance mechanisms that limit efficacy of PARPi monotherapy. Here we show how the efficacy of PARPi in triple-negative breast cancers (TNBC) can be expanded by targeting MYC-induced oncogenic addiction. In BRCA-mutant/sporadic TNBC patients, amplification of the MYC gene is correlated with increased expression of the homologous DNA recombination enzyme RAD51 and tumors overexpressing both genes are associated with worse overall survival. Combining MYC blockade with PARPi yielded synthetic lethality in MYC-driven TNBC cells. Using the cyclin-dependent kinase inhibitor dinaciclib, which downregulates MYC expression, we found that combination with the PARPi niraparib increased DNA damage and downregulated homologous recombination, leading to subsequent downregulation of the epithelial-mesenchymal transition and cancer stem-like cell phenotypes. Notably, dinaciclib resensitized TBNC cells, which had acquired resistance to niraparib. We found that the synthetic lethal strategy employing dinaciclib and niraparib was also highly efficacious in ovarian, prostate, pancreatic, colon, and lung cancer cells. Taken together, our results show how blunting MYC oncogene addiction can leverage cancer cell sensitivity to PARPi, facilitating the clinical use of c-myc as a predictive biomarker for this treatment.Significance: Dual targeting of MYC-regulated homologous recombination and PARP-mediated DNA repair yields potent synthetic lethality in triple-negative breast tumors and other aggressive tumors characterized by MYC overexpression. Cancer Res; 78(3); 742-57. ©2017 AACR.

Cyclin E Overexpression Sensitizes Triple-Negative Breast Cancer to Wee1 Kinase Inhibition.

PURPOSE: Poor prognosis in triple-negative breast cancer (TNBC) is due to an aggressive phenotype and lack of biomarker-driven targeted therapies. Overexpression of cyclin E and phosphorylated-CDK2 are correlated with poor survival in patients with TNBC, and the absence of CDK2 desensitizes cells to inhibition of Wee1 kinase, a key cell-cycle regulator. We hypothesize that cyclin E expression can predict response to therapies, which include the Wee1 kinase inhibitor, AZD1775. EXPERIMENTAL DESIGN: Mono- and combination therapies with AZD1775 were evaluated in TNBC cell lines and multiple patient-derived xenograft (PDX) models with different cyclin E expression profiles. The mechanism(s) of cyclin E-mediated replicative stress were investigated following cyclin E induction or CRISPR/Cas9 knockout by a number of assays in multiple cell lines. RESULTS: Cyclin E overexpression (i) is enriched in TNBCs with high recurrence rates, (ii) sensitizes TNBC cell lines and PDX models to AZD1775, (iii) leads to CDK2-dependent activation of DNA replication stress pathways, and (iv) increases Wee1 kinase activity. Moreover, treatment of cells with either CDK2 inhibitors or carboplatin leads to transient transcriptional induction of cyclin E (in cyclin E-low tumors) and result in DNA replicative stress. Such drug-mediated cyclin E induction in TNBC cells and PDX models sensitizes them to AZD1775 in a sequential treatment combination strategy.Conclusions: Cyclin E is a potential biomarker of response (i) for AZD1775 as monotherapy in cyclin E-high TNBC tumors and (ii) for sequential combination therapy with CDK2 inhibitor or carboplatin followed by AZD1775 in cyclin E-low TNBC tumors.

CDK4/6 Inhibitors Sensitize Rb-positive Sarcoma Cells to Wee1 Kinase Inhibition through Reversible Cell-Cycle Arrest.

Research into the biology of soft tissue sarcomas has uncovered very few effective treatment strategies that improve upon the current standard of care which usually involves surgery, radiation, and chemotherapy. Many patients with large (>5 cm), high-grade sarcomas develop recurrence, and at that point have limited treatment options available. One challenge is the heterogeneity of genetic drivers of sarcomas, and many of these are not validated targets. Even when such genes are tractable targets, the rarity of each subtype of sarcoma makes advances in research slow. Here we describe the development of a synergistic combination treatment strategy that may be applicable in both soft tissue sarcomas as well as sarcomas of bone that takes advantage of targeting the cell cycle. We show that Rb-positive cell lines treated with the CDK4/6 inhibitor palbociclib reversibly arrest in the G1 phase of the cell cycle, and upon drug removal cells progress through the cell cycle as expected within 6-24 hours. Using a long-term high-throughput assay that allows us to examine drugs in different sequences or concurrently, we found that palbociclib-induced cell-cycle arrest poises Rb-positive sarcoma cells (SK-LMS1 and HT-1080) to be more sensitive to agents that work preferentially in S-G2 phase such as doxorubicin and Wee1 kinase inhibitors (AZD1775). The synergy between palbociclib and AZD1775 was also validated in vivo using SK-LMS1 xenografts as well as Rb-positive patient-derived xenografts (PDX) developed from leiomyosarcoma patients. This work provides the necessary preclinical data in support of a clinical trial utilizing this treatment strategy. Mol Cancer Ther; 16(9); 1751-64. ©2017 AACR.

AXL Inhibition Suppresses the DNA Damage Response and Sensitizes Cells to PARP Inhibition in Multiple Cancers.

Epithelial to mesenchymal transition (EMT) is associated with a wide range of changes in cancer cells, including stemness, chemo- and radio-resistance, and metastasis. The mechanistic role of upstream mediators of EMT has not yet been well characterized. Recently, we showed that non-small cell lung cancers (NSCLC) that have undergone EMT overexpress AXL, a receptor tyrosine kinase. AXL is also overexpressed in a subset of triple-negative breast cancers (TNBC) and head and neck squamous cell carcinomas (HNSCC), and its overexpression has been associated with more aggressive tumor behavior and linked to resistance to chemotherapy, radiotherapy, and targeted therapy. Because the DNA repair pathway is also altered in patient tumor specimens overexpressing AXL, it is hypothesized that modulation of AXL in cells that have undergone EMT will sensitize them to agents targeting the DNA repair pathway. Downregulation or inhibition of AXL directly reversed the EMT phenotype, led to decreased expression of DNA repair genes, and diminished efficiency of homologous recombination (HR) and RAD51 foci formation. As a result, AXL inhibition caused a state of HR deficiency in the cells, making them sensitive to inhibition of the DNA repair protein, PARP1. AXL inhibition synergized with PARP inhibition, leading to apoptotic cell death. AXL expression also associated positively with markers of DNA repair across TNBC, HNSCC, and NSCLC patient cohorts. IMPLICATIONS: The novel role for AXL in DNA repair, linking it to EMT, suggests that AXL can be an effective therapeutic target in combination with targeted therapy such as PARP inhibitors in several different malignancies. Mol Cancer Res; 15(1); 45-58. ©2016 AACR.

CDK4/6 and autophagy inhibitors synergistically induce senescence in Rb positive cytoplasmic cyclin E negative cancers.

Deregulation of the cell cycle machinery is a hallmark of cancer. While CDK4/6 inhibitors are FDA approved (palbociclib) for treating advanced estrogen receptor-positive breast cancer, two major clinical challenges remain: (i) adverse events leading to therapy discontinuation and (ii) lack of reliable biomarkers. Here we report that breast cancer cells activate autophagy in response to palbociclib, and that the combination of autophagy and CDK4/6 inhibitors induces irreversible growth inhibition and senescence in vitro, and diminishes growth of cell line and patient-derived xenograft tumours in vivo. Furthermore, intact G1/S transition (Rb-positive and low-molecular-weight isoform of cyclin E (cytoplasmic)-negative) is a reliable prognostic biomarker in ER positive breast cancer patients, and predictive of preclinical sensitivity to this drug combination. Inhibition of CDK4/6 and autophagy is also synergistic in other solid cancers with an intact G1/S checkpoint, providing a novel and promising biomarker-driven combination therapeutic strategy to treat breast and other solid tumours.