The National Sexually Transmitted Disease Curriculum Podcast as a Method to Increase Sexually Transmitted Infection Education for Health Care Professionals.

BACKGROUND: Podcasts are a valuable educational tool that are convenient and provide on-demand learning. We launched the National Sexually Transmitted Disease Curriculum (NSTDC) Podcast in 2020 to educate health care professionals on sexually transmitted infections with an emphasis on content from peer-reviewed literature relevant to clinical practice. METHODS: We describe the reach and usage data for 31 podcast episodes produced during the first 29 months. Information was obtained via Google Analytics, Apple Podcasts, the podcast hosting platform Buzzsprout, and the Health Professional Application for Training form for listeners who were registered on the NSTDC website. RESULTS: There were more than 21,000 downloads, with an average of 686 downloads per episode. Although 85% of downloads occurred in the United States, podcast visitors were located in 57 countries. The 3 most reported professions/disciplines were registered nurse (39.0%), advanced practice nurse (22.5%), and physician (11.3%). Forty-eight percent of visitors had a primary programmatic focus of sexually transmitted diseases, 24% HIV/AIDs, and 18% primary care. CONCLUSION: The NSTDC Podcast is a highly utilized resource for mobile and on-demand learning for health care professionals who want to expand their knowledge on sexually transmitted infections.

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

BRD4 degradation blocks expression of MYC and multiple forms of stem cell resistance in Ph+ chronic myeloid leukemia.

In most patients with chronic myeloid leukemia (CML) clonal cells can be kept under control by BCR::ABL1 tyrosine kinase inhibitors (TKI). However, overt resistance or intolerance against these TKI may occur. We identified the epigenetic reader BRD4 and its downstream-effector MYC as growth regulators and therapeutic targets in CML cells. BRD4 and MYC were found to be expressed in primary CML cells, CD34+ /CD38- leukemic stem cells (LSC), and in the CML cell lines KU812, K562, KCL22, and KCL22T315I . The BRD4-targeting drug JQ1 was found to suppress proliferation in KU812 cells and primary leukemic cells in the majority of patients with chronic phase CML. In the blast phase of CML, JQ1 was less effective. However, the BRD4 degrader dBET6 was found to block proliferation and/or survival of primary CML cells in all patients tested, including blast phase CML and CML cells exhibiting the T315I variant of BCR::ABL1. Moreover, dBET6 was found to block MYC expression and to synergize with BCR::ABL1 TKI in inhibiting the proliferation in the JQ1-resistant cell line K562. Furthermore, BRD4 degradation was found to overcome osteoblast-induced TKI resistance of CML LSC in a co-culture system and to block interferon-gamma-induced upregulation of the checkpoint antigen PD-L1 in LSC. Finally, dBET6 was found to suppress the in vitro survival of CML LSC and their engraftment in NSG mice. Together, targeting of BRD4 and MYC through BET degradation sensitizes CML cells against BCR::ABL1 TKI and is a potent approach to overcome multiple forms of drug resistance in CML LSC.

Redistribution, homing and organ-invasion of neoplastic stem cells in myeloid neoplasms.

The development of a myeloid neoplasm is a step-wise process that originates from leukemic stem cells (LSC) and includes pre-leukemic stages, overt leukemia and a drug-resistant terminal phase. Organ-invasion may occur in any stage, but is usually associated with advanced disease and a poor prognosis. Sometimes, extra-medullary organ invasion shows a metastasis-like or even sarcoma-like destructive growth of neoplastic cells in local tissue sites. Examples are myeloid sarcoma, mast cell sarcoma and localized blast phase of chronic myeloid leukemia. So far, little is known about mechanisms underlying re-distribution and extramedullary dissemination of LSC in myeloid neoplasms. In this article, we discuss mechanisms through which LSC can mobilize out of the bone marrow niche, can transmigrate from the blood stream into extramedullary organs, can invade local tissue sites and can potentially create or support the formation of local stem cell niches. In addition, we discuss strategies to interfere with LSC expansion and organ invasion by targeted drug therapies.

Leveraging E-Learning Infrastructure in Times of Rapid Change: Use of the National Sexually Transmitted Diseases Curriculum in the Era of COVID-19.

ABSTRACT: The National Sexually Transmitted Diseases Curriculum is an e-learning platform. New registrations and learning group creations in March to April 2020 were compared with previous 12-month data. Substantial increases in registrations and learning groups demonstrate that the National Sexually Transmitted Diseases Curriculum was successfully leveraged to meet rapidly shifting training needs due to the COVID-19 pandemic.

Evaluation of the National Sexually Transmitted Disease Curriculum: Reach, Utilization, and Engagement.

BACKGROUND: With increasing rates of sexually transmitted infections in the United States, there is a critical need to educate health professionals on the prevention, diagnosis, and treatment of sexually transmitted infections. The National Sexually Transmitted Disease Curriculum (NSTDC, https://www.std.uw.edu) is a free, online curriculum, funded by the Centers for Disease Control and Prevention. The purpose of this article is to evaluate the reach, utilization, and engagement of users with the curriculum. METHODS: Data on NSTDC utilization was collected for 24 months after the February 1, 2017 launch. For all users, Google Analytics was used to determine total number of users, geographic location, age and sex, and average session duration. For registered users, additional data analysis included work-role, demographics, and completion of self-study modules, check-on-learning questions, and question banks. User satisfaction was measured on a 5-point Likert scale. RESULTS: During the evaluation period, 136,270 individual users accessed the NSTDC, including 24,652 registered users. Among all registered users, 10,660 (43.2%) were registered nurses, 2810 (11.4%) physicians, 4942 (20.1%) Advanced Practice Nurses and Physician Assistants, and 6213 (25.2%) nonclinicians. Among registered users, 18,533 (75.2%) completed at least 1 module, 7898 (32.0%) completed all 7 modules, and 19,804 (80.4%) answered optional check-on-learning questions. Median satisfaction with the content was (5) very satisfied (interquartile range, 4-5). CONCLUSIONS: The NSTDC is a free, guideline-based, online curriculum with novel dual functionality that has achieved extensive reach with a broad array of health professionals who engage deeply with the material. The wide usage of NSTDC demonstrates the need for high-quality, unbiased, free content in user-focused formats.

Immunotherapy-Based Targeting and Elimination of Leukemic Stem Cells in AML and CML.

The concept of leukemic stem cells (LSC) has been developed with the idea to explain the clonal hierarchies and architectures in leukemia, and the more or less curative anti-neoplastic effects of various targeted drugs. It is now widely accepted that curative therapies must have the potential to eliminate or completely suppress LSC, as only these cells can restore and propagate the malignancy for unlimited time periods. Since LSC represent a minor cell fraction in the leukemic clone, little is known about their properties and target expression profiles. Over the past few years, several cell-specific immunotherapy concepts have been developed, including new generations of cell-targeting antibodies, antibody-toxin conjugates, bispecific antibodies, and CAR-T cell-based strategies. Whereas such concepts have been translated and may improve outcomes of therapy in certain lymphoid neoplasms and a few other malignancies, only little is known about immunological targets that are clinically relevant and can be employed to establish such therapies in myeloid neoplasms. In the current article, we provide an overview of the immunologically relevant molecular targets expressed on LSC in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In addition, we discuss the current status of antibody-based therapies in these malignancies, their mode of action, and successful examples from the field.

Identification of the Ki-1 antigen (CD30) as a novel therapeutic target in systemic mastocytosis.

The Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin lymphoma and anaplastic large-cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MCs) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MCs in advanced SM, and whether this surface structure may serve as therapeutic target in SM. As assessed by flow cytometry, CD30 was found to be expressed on the surface of neoplastic MCs in 3 of 25 patients (12%) with indolent SM, 4 of 7 patients (57%) with aggressive SM, and 4 of 7 patients (57%) with MC leukemia. The immature RAS-transformed human MC line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed MC line HMC-1.2 expressed no detectable CD30. The CD30-targeting antibody-conjugate brentuximab-vedotin inhibited proliferation in neoplastic MCs, with lower IC50 values obtained in CD30(+) MCPV-1.1 cells (10 µg/mL) compared with CD30(-) HMC-1.2 cells (>50 µg/mL). In addition, brentuximab-vedotin suppressed the engraftment of MCPV-1.1 cells in NSG mice. Moreover, brentuximab-vedotin produced apoptosis in all CD30(+) MC lines tested as well as in primary neoplastic MCs in patients with CD30(+) SM, but did not induce apoptosis in neoplastic MCs in patients with CD30(-) SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE-induced histamine release in CD30(+) MCs. Finally, brentuximab-vedotin and the KIT D816V-targeting drug PKC412 produced synergistic growth-inhibitory effects in MCPV-1.1 cells. Together, CD30 is a promising new drug target for patients with CD30(+) advanced SM.

Experiences with insecticide-treated curtains: a qualitative study in Iquitos, Peru.

BACKGROUND: Dengue is an arthropod-borne viral disease responsible for approximately 400 million infections annually; the only available method of prevention is vector control. It has been previously demonstrated that insecticide treated curtains (ITCs) can lower dengue vector infestations in and around houses. As part of a larger trial examining whether ITCs could reduce dengue transmission in Iquitos, Peru, the objective of this study was to characterize the participants' experience with the ITCs using qualitative methods. METHODS: Knowledge, attitudes, and practices (KAP) surveys (at baseline, and 9 and 27 months post-ITC distribution, with n = 593, 595 and 511, respectively), focus group discussions (at 6 and 12 months post-ITC distribution, with n = 18 and 33, respectively), and 11 one-on-one interviews (at 12 months post-distribution) were conducted with 605 participants who received ITCs as part of a cluster-randomized trial. RESULTS: Focus groups at 6 months post-ITC distribution revealed that individuals had observed their ITCs to function for approximately 3 months, after which they reported the ITCs were no longer working. Follow up revealed that the ITCs required re-treatment with insecticide at approximately 1 year post-distribution. Over half (55.3 %, n = 329) of participants at 9 months post-ITC distribution and over a third (34.8 %, n = 177) at 27 months post-ITC distribution reported perceiving a decrease in the number of mosquitoes in their home. The percentage of participants who would recommend ITCs to their family or friends in the future remained high throughout the study (94.3 %, n = 561 at 9 months and 94.6 %, n = 488 at 27 months post-distribution). When asked why, participants reported that ITCs were effective at reducing mosquitoes (81.6 and 37.8 %, at 9 and 27 months respectively), that they prevent dengue (5.7 and 51.2 %, at 9 and 27 months), that they are "beautiful" (5.9 and 3.1 %), as well as other reasons (6.9 and 2.5 %). CONCLUSION: ITCs have substantial potential for long term dengue vector control because they are liked by users, both for their perceived effectiveness and for aesthetic reasons, and because they require little proactive behavioral effort on the part of the users. Our results highlight the importance of gathering process (as opposed to outcome) data during vector control studies, without which researchers would not have become aware that the ITCs had lost effectiveness early in the trial.

Presenilin 1/γ-secretase modulates P-cadherin processing and influences cell adhesion in oral squamous cell carcinoma cell lines.

P-cadherin belongs to a family of Ca(2+)-dependent homophilic cell-cell adhesion proteins that are important for correct cellular localization and tissue integrity in the oral epithelium. P-cadherin is only expressed in the basal and suprabasal cell layers of the oral epithelium, but in advanced oral squamous cell carcinoma (OSCC), a reduced membranous and an enhanced cytoplasmic truncated P-cadherin level is observed. In this study, we investigated the impact of presenilin (PS) 1/γ-secretase on P-cadherin processing in OSCC. Western blot analyses showed an enhanced PS1 expression in OSCC cell lines and in primary oral keratinocytes (POK) isolated from primary OSCC tissue (OSCC POK) compared with POKs isolated from normal oral mucosa. Immunocytochemical stainings and co-immunoprecipitation experiments revealed a cytoplasmic colocalization and a direct interaction of P-cadherin and PS1 in OSCC POKs. Blocking of PS1/γ-secretase activity by the PS1/γ-secretase inhibitors and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, another specific γ-secretase inhibitor yielded a 100 kDa P-cadherin band on western blots of OSCC cell line extracts. Small interfering RNA knockdown of PS1 equally generated a 100 kDa P-cadherin form in OSCC POKs. Mass spectrometry analyses and experiments with the glycosylation inhibitor tunicamycin characterized the appearing 100 kDa P-cadherin band as the unglycosylated full-length form of P-cadherin. On the functional level, cell attachment assays demonstrated an enhanced cell adhesion after PS1/γ-secretase inhibition only in the transiently P-cadherin expressing OSCC cell line PCI52 but not in the PCI52 control cells. In summary, our results show that PS1/γ-secretase contributes to P-cadherin processing and to reduced cell adhesion in OSCC.

The human HECA interacts with cyclins and CDKs to antagonize Wnt-mediated proliferation and chemoresistance of head and neck cancer cells.

There is a growing evidence that the human homologue of the Drosophila headcase (HECA) plays an important role in human carcinogenesis. So far specific protein interaction partners and affected signaling pathways of HECA are still elusive. In a recent study we showed that HECA overexpression in oral squamous-cell carcinoma (OSCC) keratinocytes has tumor suppressive effects resulting in a recuperation of cell cycle control concerning the entry and progression of S-phase, G2- and M-phase. Currently, quantitative RT-PCR and immunohistochemical analysis of primary tumor tissue from OSCC patients demonstrate that HECA expression is markedly decreased compared to normal control patients with abundant HECA expression. Additionally, there is nearly no HECA expression in OSCC metastases. Here, we show that HECA expression is negatively controlled by the Wnt-pathway and TCF4, a Wnt related transcription factor, binds to the HECA promoter. Furthermore, immunocytochemistry reveals colocalization of HECA with the cyclin dependent kinase CDK9. Immunoprecipitation experiments and proximity ligation assays further reveal an interaction of HECA with CDK2, CDK9, Cyclin A and Cyclin K, a direct transcriptional target of the p53 tumor suppressor. Silencing HECA in OSCC cell lines leads to a significant increase of cell division and a markedly increased resistance against the chemotherapeutic cisplatin. On the contrary, HECA overexpressing OSCC cell lines show decreased resistance of OSCC cells against cisplatin. Therefore, HECA could be considered as future therapeutic agent against Wnt-dependent tumor progression.

P-cadherin controls the differentiation of oral keratinocytes by regulating cytokeratin 1/10 expression via C/EBP-beta-mediated signaling.

P-cadherin belongs to the family of Ca(2+)-dependent homophilic glycosylated cell adhesion molecules. In the normal oral epithelium it shows a strong expression in the basal cell layer which gradually decreases in the suprabasal cell layers. The exact role of P-cadherin during the development and homeostasis of the oral epithelium has not been elucidated, yet. Here, we show for the first time that P-cadherin controls differentiation by regulating cytokeratin (CK) 1/10 expression in primary oral keratinocytes (POK) from normal, but interestingly not in POKs from oral squamous cell carcinoma (OSCC) tissue. SiRNA knockdown of P-cadherin in normal POKs revealed a strong upregulation of CK1/10 expression on mRNA and protein level. In contrast, E-cadherin knockdown in normal oral keratinocytes did not show any influence on CK1/10 expression. Moreover, in comparison with normal control keratinocytes normal oral keratinocytes with reduced P-cadherin expression displayed an enhanced expression and a stronger nuclear staining of C/EBP-beta, a well-known regulator of CK1/10 expression in keratinocytes. Furthermore, after P-cadherin knockdown in normal POKs the promoter activity of a C/EBP-responsive luciferase construct was significantly higher than in normal POKs with regular P-cadherin expression. Additionally, we noticed a proliferation advantage in normal oral keratinocytes in contrast to keratinocytes with diminished P-cadherin expression. However, the inverted effect was seen in tumor derived primary oral keratinocytes. In summary, we show that P-cadherin contributes to the keratinocyte differentiation in the oral epithelium by influencing the CK1 and CK10 expression via C/EBP-beta-mediated signaling in normal but not in tumor derived oral keratinocytes from OSCC patients.

Antineoplastic efficacy profiles of avapritinib and nintedanib in KIT D816V+ systemic mastocytosis: a preclinical study.

Systemic mastocytosis (SM) is a hematopoietic neoplasm with a complex pathology and a variable clinical course. Clinical symptoms result from organ infiltration by mast cells (MC) and the effects of pro-inflammatory mediators released during MC activation. In SM, growth and survival of MC are triggered by various oncogenic mutant-forms of the tyrosine kinase KIT. The most prevalent variant, D816V, confers resistance against various KIT-targeting drugs, including imatinib. We examined the effects of two novel promising KIT D816V-targeting drugs, avapritinib and nintedanib, on growth, survival, and activation of neoplastic MC and compared their activity profiles with that of midostaurin. Avapritinib was found to suppress growth of HMC-1.1 cells (KIT V560G) and HMC-1.2 cells (KIT V560G + KIT D816V) with comparable IC50 values (0.1-0.25 µM). In addition, avapritinib was found to inhibit the proliferation of ROSAKIT WT cells, (IC50: 0.1-0.25 µM), ROSAKIT D816V cells (IC50: 1-5 µM), and ROSAKIT K509I cells (IC50: 0.1-0.25 µM). Nintedanib exerted even stronger growth-inhibitory effects in these cells (IC50 in HMC-1.1: 0.001-0.01 µM; HMC-1.2: 0.25-0.5 µM; ROSAKIT WT: 0.01-0.1 µM; ROSAKIT D816V: 0.5-1 µM; ROSAKIT K509I: 0.01-0.1 µM). Avapritinib and nintedanib also suppressed the growth of primary neoplastic cells in most patients with SM examined (avapritinib IC50: 0.5-5 µM; nintedanib IC50: 0.1-5 µM). Growth-inhibitory effects of avapritinib and nintedanib were accompanied by signs of apoptosis and decreased surface expression of the transferrin receptor CD71 in neoplastic MC. Finally, we were able to show that avapritinib counteracts IgE-dependent histamine secretion in basophils and MC in patients with SM. These effects of avapritinib may explain the rapid clinical improvement seen during treatment with this KIT inhibitor in patients with SM. In conclusion, avapritinib and nintedanib are new potent inhibitors of growth and survival of neoplastic MC expressing various KIT mutant forms, including D816V, V560G, and K509I, which favors the clinical development and application of these new drugs in advanced SM. Avapritinib is of particular interest as it also blocks mediator secretion in neoplastic MC.

CAR virus receptor mediates erythroid differentiation and migration and is downregulated in MDS.

Myelodysplastic syndromes (MDS) are myeloid neoplasms presenting with dysplasia in the bone marrow (BM) and peripheral cytopenia. In most patients anemia develops. We screened for genes that are expressed abnormally in erythroid progenitor cells (EP) and contribute to the pathogenesis of MDS. We found that the Coxsackie-Adenovirus receptor (CAR = CXADR) is markedly downregulated in CD45low/CD105+ EP in MDS patients compared to control EP. Correspondingly, the erythroblast cell lines HEL, K562, and KU812 stained negative for CAR. Lentiviral transduction of the full-length CXADR gene into these cells resulted in an increased expression of early erythroid antigens, including CD36, CD71, and glycophorin A. In addition, CXADR-transduction resulted in an increased migration against a serum protein gradient, whereas truncated CXADR variants did not induce expression of erythroid antigens or migration. Furthermore, conditional knock-out of Cxadr in C57BL/6 mice resulted in anemia and erythroid dysplasia. Finally, decreased CAR expression on EP was found to correlate with high-risk MDS and decreased survival. Together, CAR is a functionally relevant marker that is down-regulated on EP in MDS and is of prognostic significance. Decreased CAR expression may contribute to the maturation defect and altered migration of EP and thus their pathologic accumulation in the BM in MDS.

Direct and Indirect Effects of a Project ECHO Longitudinal Clinical Tele-Mentoring Program on Viral Suppression for Persons With HIV: A Population-Based Analysis.

BACKGROUND: Project Extension for Community Health Outcomes (ECHO) aims to connect community providers to academic specialists, deliver longitudinal clinical mentorship and case consultations, plus encourage dissemination of knowledge and resources. The impact on outcomes for persons with HIV (PWH) is uncertain. SETTING: PWH in Washington and Oregon outside of the Seattle and Portland metro areas, January 2011 to March 2018. METHODS: Using viral load (VL) surveillance data, we assessed difference in the percentage of PWH who were virally suppressed among PWH whose providers participated versus did not participate in Project ECHO. Analyses included multiple mixed-effects regression models, adjusting for time and for patient, provider, and clinic characteristics. RESULTS: Based on 65,623 VL results, Project ECHO participation was associated with an increase in the percentage of patients with VL suppression (13.7 percentage points greater; P < 0.0001), although the effect varied by estimated provider PWH patient volume. The difference was 14.7 percentage points ( P < 0.0001) among patients of providers who order <20 VL's/quarter and 2.3 and -0.6 percentage points among patients of providers who order 20-40 or >40 VL's/quarter, respectively ( P > 0.5). The magnitude of difference in VL suppression was associated with the number of sessions attended. Among patients of lower-volume providers who did not participate, VL suppression was 6.2 percentage points higher if providers worked in a clinic where another provider did participate ( P < 0.0001). CONCLUSION: Project ECHO is associated with improvement in VL suppression for PWH whose providers participate or work in the same clinic system as a provider who participates, primarily because of benefits for patients of lower-volume providers.

CDK4/CDK6 Inhibitors Synergize with Midostaurin, Avapritinib, and Nintedanib in Inducing Growth Inhibition in KIT D816V+ Neoplastic Mast Cells.

In most patients with advanced systemic mastocytosis (AdvSM), neoplastic mast cells (MC) express KIT D816V. However, despite their disease-modifying potential, KIT D816V-targeting drugs, including midostaurin and avapritinib, may not produce long-term remissions in all patients. Cyclin-dependent kinase (CDK) 4 and CDK6 are promising targets in oncology. We found that shRNA-mediated knockdown of CDK4 and CDK6 results in growth arrest in the KIT D816V+ MC line HMC-1.2. The CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib suppressed the proliferation in primary neoplastic MC as well as in all HMC-1 and ROSA cell subclones that were examined. Abemaciclib was also found to block growth in the drug-resistant MC line MCPV-1, whereas no effects were seen with palbociclib and ribociclib. Anti-proliferative drug effects on MC were accompanied by cell cycle arrest. Furthermore, CDK4/CDK6 inhibitors were found to synergize with the KIT-targeting drugs midostaurin, avapritinib, and nintedanib in inducing growth inhibition and apoptosis in neoplastic MCs. Finally, we found that CDK4/CDK6 inhibitors induce apoptosis in CD34+/CD38- stem cells in AdvSM. Together, CDK4/CDK6 inhibition is a potent approach to suppress the growth of neoplastic cells in AdvSM. Whether CDK4/CDK6 inhibitors can improve clinical outcomes in patients with AdvSM remains to be determined in clinical trials.

Slit-2 facilitates interaction of P-cadherin with Robo-3 and inhibits cell migration in an oral squamous cell carcinoma cell line.

Slits are a group of secreted glycoproteins that act as molecular guidance cues in cellular migration. Recently, several studies demonstrated that Slit-2 can operate as candidate tumour suppressor protein in various tissues. In this study, we show Slit-2 expression in basal cell layers of normal oral mucosa colocalized with P-cadherin expression. In contrast, there is a loss of Slit-2 and P-cadherin expression in mucosa of oral squamous cell carcinoma (OSCC). Our in vitro investigations reveal a correlation of P-cadherin and Slit-2 expression: OSCC cells with induced P-cadherin expression (PCI52_PC) display an increased Slit-2 expression. However, abrogating P-cadherin function with a function-blocking antibody decreases Slit-2 secretion confirming a direct link between P-cadherin and Slit-2. Moreover, experiments with OSCC cells show that Slit-2 interferes with a Wnt related signalling pathway, which in turn affects Slit-2 expression in a feedback loop. Functionally, transwell migration assays demonstrate a Slit-2 dose-dependent decrease of PCI52_PC cell migration. However, there is no influence on migration in mock control cells. Responsible for this migration block might be an interaction of P-cadherin with Roundabout (Robo)-3, a high affinity receptor of Slit-2. Indeed, proximity ligation assays exhibit P-cadherin/Robo-3 interactions on PCI52_PC cells. Additionally, we detect a modulation of this interaction by addition of recombinant Slit-2. Down-regulation of Robo-3 expression via small interfering RNA neutralizes Slit-2 induced migration block in PCI52_PC cells. In summary, our experiments show antitumorigenic effects of Slit-2 on P-cadherin expressing OSCC cells supposedly via modulation of Robo-3 interaction.

Degradation of BRD4 - a promising treatment approach not only for hematologic but also for solid cancer.

Bromodomain (BRD) and extra-terminal (BET) proteins are epigenetic readers that regulate gene expression and promote cancer evolution. Pharmacological inactivation of BRD4 has recently been introduced as a promising anti-neoplastic approach that targets MYC oncogene expression. However, resistance against BRD4-targeting drugs has been described. We compared the efficacy of the small-molecule-type BET BRD inhibitor JQ1 with the recently developed BET protein degraders dBET1 and dBET6 in colon, breast, melanoma, ovarian, lung and prostate cancer cell lines. As determined by qPCR, all BRD4 targeting drugs dose-dependently decreased MYC expression, with dBET6 introducing the strongest downregulation of MYC. This correlated with the anti-proliferative activity of these drugs, which was at least one order of magnitude higher for dBET6 (IC50 0.001-0.5 µM) than for dBET1 or JQ1 (IC50 0.5-5 µM). Interestingly, when combined with commonly used cytotoxic therapeutics, dBET6 was found to promote anti-neoplastic effects and to counteract chemoresistance in most cancer cell lines. Moreover, JQ1 and both BET degraders strongly downregulated baseline and interferon-gamma induced expression of the immune checkpoint molecule PD-L1 in all cancer cell lines. Together, our data suggest that dBET6 outperforms first-generation BRD4 targeting drugs like dBET1 and JQ1, and decreases chemoresistance and immune resistance of cancer.

PI3-kinase inhibition as a strategy to suppress the leukemic stem cell niche in Ph+ chronic myeloid leukemia.

Recent data suggest that the disease-associated microenvironment, known as the leukemic stem cell (LSC) niche, is substantially involved in drug resistance of LSC in BCR-ABL1+ chronic myeloid leukemia (CML). Attacking the LSC niche in CML may thus be an effective approach to overcome drug resistance. We have recently shown that osteoblasts are a major site of niche-mediated LSC resistance against second- and third-generation tyrosine kinase inhibitors (TKI) in CML. In the present study, we screened for drugs that are capable of suppressing the growth and viability of osteoblasts and/or other niche cells and can thereby overcome TKI resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells, and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC50: 0.05 µM; copanlisib IC50: 0.05 µM), the osteoblast cell line CAL-72 (BEZ235 IC50: 0.5 µM; copanlisib IC50: 1 µM), primary umbilical vein-derived endothelial cells (BEZ235 IC50: 0.5 µM; copanlisib IC50: 0.5 µM), and the vascular endothelial cell line HMEC-1 (BEZ235 IC50: 1 µM; copanlisib IC50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. Furthermore, we show that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing proliferation and survival of osteoblasts and endothelial cells. Finally, BEZ235 and copanlisib were found to overcome osteoblast-mediated resistance against nilotinib and ponatinib in K562 cells, KU812 cells and primary CD34+/CD38- CML LSC. Together, targeting osteoblastic niche cells through PI3K inhibition may be a new effective approach to overcome niche-induced TKI resistance in CML. Whether this approach can be translated into clinical application and can counteract drug resistance of LSC in patients with CML remains to be determined in clinical trials.

Secondary basophilic leukemia in Ph-negative myeloid neoplasms: A distinct subset with poor prognosis.

During progression of myeloid neoplasms, the basophil compartment may expand substantially and in some of these patients, a basophilic leukemia is diagnosed. In patients with Ph-chromosome+ chronic myeloid leukemia, acceleration of disease is typically accompanied by marked basophilia. In other myeloid neoplasms, secondary leukemic expansion of basophils is rarely seen. We report on 5 patients who suffered from a myelodysplastic syndrome, myeloproliferative neoplasm, or acute leukemia and developed a massive expansion of basophils during disease progression. In 4 of 5 patients, peripheral blood basophil counts reached 40%, and the diagnosis "secondary basophilic leukemia" was established. As assessed by flow cytometry, neoplastic basophils expressed CD9, CD18, CD25, CD33, CD63, PD-L1, CD123, and CLL-1. In addition, basophils were found to display BB1 (basogranulin), 2D7, tryptase and KIT. In 4 of 5 patients the disease progressed quickly and treatment with azacitidine was started. However, azacitidine did not induce major clinical responses, and all patients died from progressive disease within 3 Y. In in vitro experiments, the patients´ cells and the basophilic leukemia cell line KU812 showed variable responses to targeted drugs, including azacitidine, venetoclax, hydroxyurea, and cytarabine. A combination of venetoclax and azacitidine induced cooperative antineoplastic effects in these cells. Together, secondary basophilic leukemia has a poor prognosis and monotherapy with azacitidine is not sufficient to keep the disease under control for longer time-periods. Whether drug combination, such as venetoclax+azacitidine, can induce better outcomes in these patients remains to be determined in future clinical studies.