CD73 Is an Immunometabolic Biomarker of Poor Prognosis in Patients With Primary Cutaneous Squamous Cell Carcinoma and Hematologic Malignancy.

BACKGROUND: Impaired immunity may drive the increased incidence and aggression of cutaneous squamous cell carcinoma (cSCC) in patients with hematologic malignancy; however, precise mechanisms and prognostic biomarkers remain undefined. CD73 maintains elevated immunosuppressive adenosine levels and is associated with poor prognosis in several tumor microenvironments. OBJECTIVE: Identify poor outcome biomarkers in patients with cSCC and hematologic malignancy. MATERIALS AND METHODS: Differentially expressed genes in tumors from patients with hematologic malignancy experiencing good (n = 8) versus poor (n = 7) outcomes were identified by NanoString analysis. Results were validated at the protein level using CD73 immunohistochemistry in cSCC patients with (n = 38) and without (n = 29) hematologic malignancy. RESULTS: Forty-eight genes were differentially expressed in tumors from patients with hematologic malignancy experiencing good versus poor outcomes. CD73 gene expression was >2-fold higher in patients with poor versus good outcomes or normal skin. Significantly increased CD73 protein levels were observed in cSCC tumors with poor versus good outcomes from patients with hematologic malignancies (p < .01), whereas no differences were noted in tumors with poor versus good outcomes from patients without hematologic malignancies (p = .49). CONCLUSION: CD73 is highly expressed in poor prognosis cSCC from patients with hematologic malignancy and may represent a useful biomarker and potential therapeutic target.

Defining and quantifying histopathologic risk factors for regional and distant metastases in a large cohort of vulvar squamous cell carcinomas.

BACKGROUND: Vulvar squamous cell carcinoma (vSCC) is a rare tumor with a good prognosis when treated at a localized stage. However, once regional/distant metastasis occurs, vSCC can be rapidly fatal. Thus, it is important to identify tumor prognostic features so that high-risk cases can be prioritized for further diagnostic workup and treatment. OBJECTIVE: To estimate the risk of regional/distant metastasis at presentation and sentinel lymph node status for vSCC based on histopathologic characteristics. METHODS: A retrospective cohort study of 15,188 adult vSCC cases from the National Cancer Database diagnosed from 2012 to 2019. RESULTS: We provide specific estimates of the risk of clinically positive nodes and metastatic disease at presentation and sentinel lymph node positivity according to tumor size, moderate/poor tumor differentiation, and lymph-vascular invasion. These histopathologic factors were all significantly associated with the tested clinical outcomes in a multivariable analysis. Moderate (hazard ratio, 1.190; P < .001) and poor differentiation (hazard ratio, 1.204; P < .001) and lymph-vascular invasion (hazard ratio, 1.465; P < .001) were also associated with significantly poorer overall survival. LIMITATIONS: Data on disease-specific survival not available in the data set. CONCLUSIONS: We demonstrate the association of the histopathologic characteristics of vSCC with clinically important outcomes. These data may provide individualized information when discussing diagnostic/treatment recommendations, particularly regarding sentinel lymph node biopsy. These data may also guide future staging and risk stratification efforts for vSCC.

Assessing Rates of Positive Surgical Margins After Standard Excision of Vulvar Melanomas.

BACKGROUND: Vulvar melanoma is a rare malignancy with frequent recurrence and poor prognosis. National guidelines recommend wide local excision of these tumors with allowances for narrower margins for anatomic and functional limitations, which are common on specialty sites. There is presently a lack of data of margin positivity after standard excision of vulvar melanomas. OBJECTIVE: We aim to evaluate the rate of positive margins after standard excision of vulvar melanomas. MATERIALS AND METHODS: Retrospective cohort study of surgically excised vulvar melanomas from the NCDB diagnosed from 2004 to 2019. RESULTS: We identified a total of 2,226 cases. Across surgical approaches and tumor stages, 17.2% (Standard Error [SE]: 0.8%) of cases had positive surgical margins. Among tumor stages, T4 tumors were most commonly excised with positive margins (22.9%, SE: 1.5%). On multivariable survival analysis, excision with positive margins was associated with significantly poorer survival (Hazard Ratio 1.299, p = .015). CONCLUSION: We find that positive margin rates after standard excision of vulvar malignancies are higher than for other specialty site melanomas. Our data suggest that use of surgical approaches with complete margin assessment may improve local control and functional outcomes for patients with vulvar melanoma as they have for patients with other specialty site melanomas.

Evaluating Rates of Positive Margins After Standard Excision of Cutaneous Adnexal Malignancies.

BACKGROUND: It is recommended to excise adnexal neoplasms with standard local excision or Mohs micrographic surgery (MMS), although many occur on high-risk sites such as the head and neck (H&N) and exhibit subclinical extension. Minimal evidence exists on the efficacy of standard excisions for these tumors. OBJECTIVE: To evaluate the rate of positive surgical margins after standard excision of adnexal tumors. METHODS: Retrospective cohort study of cutaneous adnexal malignancies from the National Cancer Database diagnosed from 2004 to 2019. RESULTS: The authors identified a total of 4,402 cases treated with standard excision. Tumors on the H&N were approximately twice as likely as those on the trunk and extremities (T&E) to be excised with positive margins (odds ratio 2.146, p < .001), with the highest estimated rate for eccrine adenocarcinoma (12.1%, SE: 2.3%). The subtype with the highest positive margin rate on the T&E was microcystic adnexal carcinoma (8.0%, SE: 2.9). Positive margins were associated with poorer overall survival on multivariable survival analysis (hazard ratio 1.299, p = .015). CONCLUSION: The authors present subtype- and site-specific positive margin rates for adnexal tumors treated with standard excision, which suggest that tumors on the H&N and some T&E subtypes, should be considered for MMS.

Personalization of cancer treatment using predictive simulation.

BACKGROUND: The personalization of cancer treatments implies the reconsideration of a one-size-fits-all paradigm. This move has spawned increased use of next generation sequencing to understand mutations and copy number aberrations in cancer cells. Initial personalization successes have been primarily driven by drugs targeting one patient-specific oncogene (e.g., Gleevec, Xalkori, Herceptin). Unfortunately, most cancers include a multitude of aberrations, and the overall impact on cancer signaling and metabolic networks cannot be easily nullified by a single drug. METHODS: We used a novel predictive simulation approach to create an avatar of patient cancer cells using point mutations and copy number aberration data. Simulation avatars of myeloma patients were functionally screened using various molecularly targeted drugs both individually and in combination to identify drugs that are efficacious and synergistic. Repurposing of drugs that are FDA-approved or under clinical study with validated clinical safety and pharmacokinetic data can provide a rapid translational path to the clinic. High-risk multiple myeloma patients were modeled, and the simulation predictions were assessed ex vivo using patient cells. RESULTS: Here, we present an approach to address the key challenge of interpreting patient profiling genomic signatures into actionable clinical insights to make the personalization of cancer therapy a practical reality. Through the rational design of personalized treatments, our approach also targets multiple patient-relevant pathways to address the emergence of single therapy resistance. Our predictive platform identified drug regimens for four high-risk multiple myeloma patients. The predicted regimes were found to be effective in ex vivo analyses using patient cells. CONCLUSIONS: These multiple validations confirm this approach and methodology for the use of big data to create personalized therapeutics using predictive simulation approaches.

High-volume facilities are significantly more likely to use guideline-adherent systemic immunotherapy for metastatic Merkel cell carcinoma: implications for cancer care regionalization.

Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer with a high rate of mortality. While still relatively rare, the incidence of MCC has been rapidly rising in the US and around the world. Since 2017, two immunotherapeutic drugs, avelumab and pembrolizumab, have been FDA-approved for the treatment of metastatic MCC and have revolutionized outcomes for MCC. However, real-world outcomes can differ from clinical trial data, and the adoption of novel therapeutics can be gradual. We aimed to characterize the treatment practices and outcomes of patients with metastatic MCC across the US. A retrospective cohort study of adult cases of MCC in the National Cancer Database diagnosed from 2004 to 2019 was performed. Multivariable logistic regressions to determine the association of a variety of patient, tumor, and system factors with likelihood of receipt of systemic therapies were performed. Univariate Kaplan-Meier and multivariable Cox survival regressions were performed. We identified 1017 cases of metastatic MCC. From 2017 to 2019, 54.2% of patients received immunotherapy. This increased from 45.1% in 2017 to 63.0% in 2019. High-volume centers were significantly more likely to use immunotherapy (odds ratio 3.235, p = 0.002). On univariate analysis, patients receiving systemic immunotherapy had significantly improved overall survival (p < 0.001). One-, 3-, and 5-year survival was 47.2% (standard error [SE] 1.8%), 21.8% (SE 1.5%), and 16.5% (SE 1.4%), respectively, for patients who did not receive immunotherapy versus 62.7% (SE 3.5%), 34.4% (SE 3.9%), and 23.6% (SE 4.4%), respectively, for those who did (Fig. 1). In our multivariable survival regression, receipt of immunotherapy was associated with an approximately 35% reduction in hazard of death (hazard ratio 0.665, p < 0.001; 95% CI 0.548-0.808). Our results demonstrate that the real-world survival advantage of immunotherapy for metastatic MCC is similar to clinical trial data. However, many patients with metastatic disease did not receive this guideline-recommended therapy in our most recent study year, and use of immunotherapy is higher at high-volume centers. This suggests that regionalization of care to high-volume centers or dissemination of their practices, may ultimately improve patient survival.

XIAP downregulation accompanies mebendazole growth inhibition in melanoma xenografts.

Mebendazole (MBZ) was identified as a promising therapeutic on the basis of its ability to induce apoptosis in melanoma cell lines through a B-cell lymphoma 2 (BCL2)-dependent mechanism. We now show that in a human xenograft melanoma model, oral MBZ is as effective as the current standard of care temozolomide in reducing tumor growth. Inhibition of melanoma growth in vivo is accompanied by phosphorylation of BCL2 and decreased levels of X-linked inhibitor of apoptosis (XIAP). Reduced expression of XIAP on treatment with MBZ is partially mediated by its proteasomal degradation. Furthermore, exposure of melanoma cells to MBZ promotes the interaction of SMAC/DIABLO with XIAP, thereby alleviating XIAP's inhibition on apoptosis. XIAP expression on exposure to MBZ is indicative of sensitivity to MBZ as MBZ-resistant cells do not show reduced levels of XIAP after treatment. Resistance to MBZ can be reversed partially by siRNA knockdown of cellular levels of XIAP. Our data indicate that MBZ is a promising antimelanoma agent on the basis of its effects on key antiapoptotic proteins.

Overall Survival After Mohs Surgery for Early-Stage Merkel Cell Carcinoma.

Importance: Merkel cell carcinoma (MCC) is a rare cutaneous malignant neoplasm with increasing incidence and high mortality. Although it is accepted that the optimal treatment for localized tumors is surgical, the data surrounding the optimal surgical approach are mixed, and current National Comprehensive Cancer Network guidelines state that Mohs micrographic surgery (MMS) and wide local excision (WLE) can both be used. The current National Comprehensive Cancer Network guidelines do not advocate a preference for MMS or WLE and suggest that they can be used interchangeably. Objective: To evaluate the association of surgical approach with overall survival after excision of localized T1/T2 MCC. Design, Setting, and Participants: This retrospective cohort study used the National Cancer Database to assess adults with T1/T2 MCC who were diagnosed between January 1, 2004, and December 31, 2018, with pathologically confirmed, negative regional lymph nodes and treated with surgery. The National Cancer Database includes all reportable cases from Commission on Cancer-accredited facilities. Data analysis was performed from October 2022 to May 2023. Exposure: Surgical approach. Main Outcomes and Measures: Overall survival. Results: A total of 2313 patients (mean [SD] age, 71 [10.6] years; 1340 [57.9%] male) were included in the study. Excision with MMS had the best unadjusted survival, with mean (SE) survival rates of 87.4% (3.4%) at 3 years, 84.5% (3.9%) at 5 years, and 81.8% (4.6%) at 10 years vs 86.1% (0.9%) at 3 years, 76.9% (1.2%) at 5 years, and 60.9% (2.0%) at 10 years for patients treated with WLE. Patients treated with narrow-margin excision had similar survival as those treated with WLE, with mean (SE) survival rates of 84.8% (1.4%) at 3 years, 78.3% (1.7%) at 5 years, and 60.8% (3.6%) at 10 years. On multivariable survival analysis, excision with MMS was associated with significantly improved survival compared with WLE (hazard ratio, 0.59; 95% CI, 0.36-0.97; P = .04). High-volume MCC centers were significantly more likely to use MMS over WLE compared with other centers (odds ratio, 1.99; 95% CI, 1.63-2.44; P < .001). Conclusions and Relevance: In this cohort study, the use of MMS (compared with WLE) was associated with significantly improved survival for patients with localized MCC with pathologically confirmed negative lymph nodes treated with surgery. These data suggest that Mohs surgery may provide a more effective treatment for MCC primary tumors than conventional WLE, although the lack of randomization and potential for selection bias in this study highlight the need for future prospective work evaluating this issue.

Self-supervised artificial intelligence predicts recurrence, metastasis and disease specific death from primary cutaneous squamous cell carcinoma at diagnosis.

Primary cutaneous squamous cell carcinoma (cSCC) is responsible for ~10,000 deaths annually in the United States. Stratification of risk of poor outcome (PO) including recurrence, metastasis and disease specific death (DSD) at initial biopsy would significantly impact clinical decision-making during the initial post operative period where intervention has been shown to be most effective. In this multi-institutional study, we developed a state-of-the-art self-supervised deep-learning approach with interpretability power and demonstrated its ability to predict poor outcomes of cSCCs at the time of initial biopsy. By highlighting histomorphological phenotypes, our approach demonstrates that poor differentiation and deep invasion correlate with poor prognosis. Our approach is particularly efficient at defining poor outcome risk in Brigham and Women's Hospital (BWH) T2a and American Joint Committee on Cancer (AJCC) T2 cSCCs. This bridges a significant gap in our ability to assess risk among T2a/T2 cSCCs and may be useful in defining patients at highest risk of poor outcome at the time of diagnosis. Early identification of highest-risk patients could signal implementation of more stringent surveillance, rigorous diagnostic work up and identify patients who might best respond to early postoperative adjunctive treatment.

Mitochondrial DNA oxidative damage and mutagenesis in Saccharomyces cerevisiae.

Mutation of human mitochondrial DNA (mtDNA) has been linked to maternally inherited neuromuscular disorders and is implicated in more common diseases such as cancer, diabetes, and Parkinson's disease. Mutations in mtDNA also accumulate with age and are therefore believed to contribute to aging and age-related pathology. Housed within the mitochondrial matrix, mtDNA encodes several of the proteins involved in the production of ATP via the process of oxidative phosphorylation, which involves the flow of high-energy electrons through the electron transport chain (ETC). Because of its proximity to the ETC, mtDNA is highly vulnerable to oxidative damage mediated by reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals that are constantly produced by this system. Therefore, it is important to be able to measure oxidative mtDNA damage under normal physiologic conditions and during environmental or disease-associated stress. The budding yeast, Saccharomyces cerevisiae, is a facile and informative model system in which to study such mtDNA oxidative damage because it is a unicellular eukaryotic facultative anaerobe that is conditionally dependent on mitochondrial oxidative phosphorylation for viability. Here, we describe methods for quantifying oxidative mtDNA damage and mutagenesis in S. cerevisiae, several of which could be applied to the development of similar assays in mammalian cells and tissues. These methods include measuring the number of point mutations that occur in mtDNA with the erythromycin resistance assay, quantifying the amount of oxidative DNA damage utilizing a modified Southern blot assay, and measuring mtDNA integrity with the "petite induction" assay.

Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae.

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins.

The mitochondrial genome is a significant target of exogenous and endogenous genotoxic agents; however, the determinants that govern this susceptibility and the pathways available to resist mitochondrial DNA (mtDNA) damage are not well characterized. Here we report that oxidative mtDNA damage is elevated in strains lacking Ntg1p, providing the first direct functional evidence that this mitochondrion-localized, base excision repair enzyme functions to protect mtDNA. However, ntg1 null strains did not exhibit a mitochondrial respiration-deficient (petite) phenotype, suggesting that mtDNA damage is negotiated by the cooperative actions of multiple damage resistance pathways. Null mutations in ABF2 or PIF1, two genes implicated in mtDNA maintenance and recombination, exhibit a synthetic-petite phenotype in combination with ntg1 null mutations that is accompanied by enhanced mtDNA point mutagenesis in the corresponding double-mutant strains. This phenotype was partially rescued by malonic acid, indicating that reactive oxygen species generated by the electron transport chain contribute to mitochondrial dysfunction in abf2 Delta strains. In contrast, when two other genes involved in mtDNA recombination, CCE1 and NUC1, were inactivated a strong synthetic-petite phenotype was not observed, suggesting that the effects mediated by Abf2p and Pif1p are due to novel activities of these proteins other than recombination. These results document the existence of recombination-independent mechanisms in addition to base excision repair to cope with oxidative mtDNA damage in Saccharomyces cerevisiae. Such systems are likely relevant to those operating in human cells where mtDNA recombination is less prevalent, validating yeast as a model system in which to study these important issues.

Differential involvement of the related DNA helicases Pif1p and Rrm3p in mtDNA point mutagenesis and stability.

With the exception of base excision repair, conserved pathways and mechanisms that maintain mitochondrial genome stability have remained largely undelineated. In the budding yeast, Saccharomyces cerevisiae, Pif1p is a unique DNA helicase that is localized both to the nucleus and mitochondria, where it is involved in maintaining DNA integrity. We previously elucidated a role for Pif1p in oxidative mtDNA damage resistance that appears to be distinct from its postulated function in mtDNA recombination. Strains lacking Pif1p (pif1Delta) exhibit an increased rate of formation of petite mutants (an indicator of mtDNA instability) and elevated mtDNA point mutagenesis. Here we show that deletion of the RRM3 gene, which encodes a DNA helicase closely related to Pif1p, significantly rescues the petite-induction phenotype of a pif1Delta strain. However, suppression of this phenotype was not accompanied by a corresponding decrease in mtDNA point mutagenesis. Instead, deletion of RRM3 alone resulted in an increase in mtDNA point mutagenesis that was synergistic with that caused by a pif1Delta mutation. In addition, we found that over-expression of RNR1, encoding a large subunit of ribonucleotide reductase (RNR), rescued the petite-induction phenotype of a pif1Delta mutation to a similar extent as deletion of RRM3. This, coupled to our finding that the Rad53p protein kinase is phosphorylated in the rrm3Delta pif1Delta double-mutant strain, leads us to conclude that one mechanism whereby deletion of RRM3 influences mtDNA stability is by modulating mitochondrial deoxynucleoside triphosphate pools. We propose that this is accomplished by signaling through the conserved Mec1/Rad53, S-phase checkpoint pathway to induce the expression and activity of RNR. Altogether, our results define a novel role for Rrm3p in mitochondrial function and indicate that Pif1p and Rrm3p influence a common process (or processes) involved in mtDNA replication, repair, or stability.

Identification of agents that promote endoplasmic reticulum stress using an assay that monitors luciferase secretion.

Disruption of protein processing in the secretory pathway is a measurable hallmark of endoplasmic reticulum (ER) stress. Activation of ER stress-mediated pathways has been implicated in numerous diseases, including cancer. To identify agents that induce ER stress, we established a screen for compounds that reduce secretion of the reporter protein Gaussia luciferase (GLUC). Given the clinically validated importance of targeting ER stress-mediated pathways in the treatment of multiple myeloma (MM), we used this hematological malignancy as a model for validating our screening system. From a screen of 2000 marketed drugs and natural compounds in KMS11 and ARP1 MM cells, we identified 97 agents that reduced GLUC secretion in both cell lines by at least 30%. To confirm inducers of ER stress, we applied a secondary screen that assessed splicing of the unfolded protein response (UPR) transcription factor XBP1. One agent, theaflavin-3,3'-digallate (TF-3), was chosen based on its history of safe human consumption and further validated through studies of ER stress-related pathways, including the UPR and apoptosis. Given these promising results, this screen could be a useful tool to identify agents targeting ER stress-related mechanisms in other cellular systems wherein ER stress plays a role in disease etiology.

Predictive simulation approach for designing cancer therapeutic regimens with novel biological mechanisms.

Introduction Ursolic acid (UA) is a pentacyclic triterpene acid present in many plants, including apples, basil, cranberries, and rosemary. UA suppresses proliferation and induces apoptosis in a variety of tumor cells via inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Given that single agent therapy is a major clinical obstacle to overcome in the treatment of cancer, we sought to enhance the anti-cancer efficacy of UA through rational design of combinatorial therapeutic regimens that target multiple signaling pathways critical to carcinogenesis. Methodology Using a predictive simulation-based approach that models cancer disease physiology by integrating signaling and metabolic networks, we tested the effect of UA alone and in combination with 100 other agents across cell lines from colorectal cancer, non-small cell lung cancer and multiple myeloma. Our predictive results were validated in vitro using standard molecular assays. The MTT assay and flow cytometry were used to assess cellular proliferation. Western blotting was used to monitor the combinatorial effects on apoptotic and cellular signaling pathways. Synergy was analyzed using isobologram plots. Results We predictively identified c-Jun N-terminal kinase (JNK) as a pathway that may synergistically inhibit cancer growth when targeted in combination with NFκB. UA in combination with the pan-JNK inhibitor SP600125 showed maximal reduction in viability across a panel of cancer cell lines, thereby corroborating our predictive simulation assays. In HCT116 colon carcinoma cells, the combination caused a 52% reduction in viability compared with 18% and 27% for UA and SP600125 alone, respectively. In addition, isobologram plot analysis reveals synergy with lowered doses of the drugs in combination. The combination synergistically inhibited proliferation and induced apoptosis as evidenced by an increase in the percentage sub-G1 phase cells and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Combination treatment resulted in a significant reduction in the expression of cyclin D1 and c-Myc as compared with single agent treatment. Conclusions Our findings underscore the importance of targeting NFκB and JNK signaling in combination in cancer cells. These results also highlight and validate the use of predictive simulation technology to design therapeutics for targeting novel biological mechanisms using existing or novel chemistry.

Sulforaphane synergistically enhances the cytotoxicity of arsenic trioxide in multiple myeloma cells via stress-mediated pathways.

Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic reticulum (ER) that is unusually susceptible to perturbations in protein synthesis. This biology is believed to account for the exquisite sensitivity of multiple myeloma (MM) to the proteasomal inhibitor bortezomib (BTZ). Despite remarkable response rates to BTZ in MM, BTZ carries the potential for serious side-effects and development of resistance. We, therefore, sought to identify therapeutic combinations that effectively disrupt proteostasis in order to provide new potential treatments for MM. We found that sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, inhibits TNFα-induced Iκß proteasomal degradation in a manner similar to BTZ. Like BTZ, sulforaphane synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent with clinical activity in MM. ATO and sulforaphane co-treatment augmented apoptotic induction as demonstrated by cleavage of caspase-3, -4 and PARP. The enhanced apoptotic response was dependent upon production of reactive oxygen species (ROS) as demonstrated by glutathione depletion and partial inhibition of the apoptotic cascade after pretreatment with the radical scavenger N-acetyl-cysteine (NAC). Combination treatment resulted in enhanced ER stress signaling and activation of the unfolded protein response (UPR), indicative of perturbation of proteostasis. Specifically, combination treatment caused elevated expression of the molecular chaperone HSP90 (heat shock protein 90) along with increased PERK (protein kinase RNA-like endoplasmic reticulum kinase) and eIF2α phosphorylation and XBP1 (X-box binding protein 1) splicing, key indicators of UPR activation. Moreover, increased splicing of XBP1 was apparent upon combination treatment compared to treatment with either agent alone. Sulforaphane in combination with ATO effectively disrupts protein homeostasis through ROS generation and induction of ER stress to culminate in inhibition of protein secretion and apoptotic induction in MM. Our results suggest that sulforaphane deserves further investigation in combination with ATO in the treatment of MM.

Antagonistic effect of small-molecule inhibitors of Wnt/ß-catenin in multiple myeloma.

BACKGROUND: Development and progression of multiple myeloma is dependent on the bone marrow (BM) microenvironment, and within the BM, a number of factors are secreted, including the Wnt ligands. Bone marrow stromal cells (BMSC) secrete Wnt ligands that activate Wnt signaling in multiple myeloma. The canonical Wnt pathway which is mediated through the transcriptional effector ß-catenin (ß-cat) is commonly de-regulated in many cancers. Cells with active ß-cat-regulated transcription (CRT) are protected against apoptosis; conversely, inhibition of CRT may prevent cell proliferation. MATERIALS AND METHODS: In this study, we tested the efficacy of recently described inhibitors of CRT (iCRTs; oxazole and thiazole) for their selective antagonistic effect on Wnt-ß-cat response in MM cells MM.1, U266, BMSC and primary BMMC obtained from patient samples (n=16). RESULTS: We demonstrated that iCRTs we used, block Wnt/ß-cat reporter activity, down regulate ß-cat expression and inhibit cell proliferation in a dose-dependent manner with an optimal dose closer to 15 µM. Our data further indicate that iCRTs do not influence the expression of the upstream components of the Wnt pathway DKK1 at the optimal dose, suggesting that iCRTs may specifically target ß-cat in MM cells. Additionally, iCRT-treatment of MM cells, co-cultured with BMSC, showed an inhibitory effect on VEGF and cell migration. CONCLUSION: This study provides the first in vitro data evaluation of newly-described iCRTs as potential Wnt-ß-cat/VEGF pathway antagonists in multiple myeloma.

Fluvastatin enhances sorafenib cytotoxicity in melanoma cells via modulation of AKT and JNK signaling pathways.

Most metastatic melanomas are refractory to current available therapy, underscoring the need to identify new effective treatments. Sorafenib, a multikinase inhibitor of the mitogen-activated protein kinase signaling pathway, showed promise in earlier stages of clinical development, but ultimately failed to demonstrate efficacy as a single agent in the treatment of melanoma. In order to enhance the efficacy of sorafenib in the treatment of melanoma, we tested over 2,000 naturally occurring compounds and marketed drugs in the presence of sorafenib in chemoresistant human melanoma cell lines also resistant to sorafenib-induced apoptosis. Of the 9 compounds identified as sorafenib sensitizers, we prioritized the cholesterol-lowering agent fluvastatin, based on its favorable pharmacokinetics and safety profile. Our results demonstrate that fluvastatin at 1 µM, a level safely achieved through oral administration, dramatically enhances the growth-inhibitory activity of clinically achievable concentrations of sorafenib in M14 and SKM-173 melanoma cells, with a 3.2- and 3.6-fold reduction in sorafenib 50% growth inhibition (GI50), respectively. Similar effects were observed for other melanoma cell lines. Combination indices analysis revealed a synergistic relationship between the two agents. Fluvastatin enhances sorafenib-mediated apoptosis as revealed through enhanced cleavage of poly (ADP-ribose) polymerase. In combination with sorafenib, fluvastatin treatment results in reduced levels of activated murine thymoma viral oncogene homolog along with enhanced levels of activated c-Jun N-terminal kinase. Sensitization to sorafenib is unique to fluvastatin, as other statins (pravastatin and simvastatin) do not enhance sorafenib-mediated growth inhibition. These promising results warrant further investigation into the clinical applicability of fluvastatin as an agent for enhancing the efficacy of sorafenib in the treatment of melanoma.