HEL Cells Research Articles

Design, synthesis and SAR study of 2-aminopyridine derivatives as potent and selective JAK2 inhibitors

The abnormal activation of JAK2 kinase is closely related to the occurrence and progression of myeloproliferative neoplasms (MPNs). At present, there is still an obvious unmet medical need for selective JAK2 inhibitors in clinic. In this paper, a class of 2-aminopyridine derivatives as potent and selective JAK2 inhibitors was obtained by combining drug design, synthesis and structure-activity relationship studies based on the previously identified lead Crizotinib. Among them, 21b exhibited high inhibitory activity against JAK2 with an IC50 of 9 nmol/L, moreover, it showed 276- and 184-fold selectivity over JAK1 and JAK3, respectively. Besides, 21b had a significant antiproliferative activity against HEL cells, and also inhibited the phosphorylation of JAK2 and its down-stream signaling pathway. These results indicated that 2-aminopyridine compound 21b had the potential to be developed as a selective JAK2 inhibitor for further study.

3118 – RUNX1 AS A REGULATOR OF MEP FATE IN RESPONSE TO CELL CYCLE DYNAMICS

Primary human megakaryocyte-erythroid progenitors (MEP) can undergo fate specification to become megakaryocytic (Mk) or erythroid (E) progenitors. Despite their importance in maintaining the blood system, their fate specification mechanism remains elusive. Based on our single cell RNA-seq data implying that RUNX1 may be a key regulator of differential gene expression in MEP fate specification, we first found that RUNX1 overexpression in MEP promotes Mk over E specification while RUNX1 inhibition causes the opposite. Because RUNX1 mRNA and protein levels did not differ between Mk vs. E, we tested whether post-translational modifications differ, and found that the level of phospho-S276 RUNX1 (pS-RUNX1) is significantly higher in Mk than E. We compared the effects on MEP fate specification of WT RUNX1 vs. RUNX1-4D (S249D, S266D, S276D and T303D mutations to mimic phosphorylation (P'n) at these sites) vs. RUNX1-4A (S249A, S266A, S276A, T303A to prevent P'n). RUNX1-4D promotes Mk specification more dramatically than WT or RUNX1-4A. To determine whether P'n of these S/T residues affects RUNX1 chromatin localization and gene expression patterns, we performed CUT&RUN of HA-tagged RUNX1 WT, 4A and 4D overexpressed in the HEL cell line, which models MEP, as well as RNA-seq of these cells. While the 3 forms of RUNX1 have similar chromatin association sites, many Mk-related genes were differentially expressed. We have published that enhancing and suppressing MEP cell cycle promotes E and Mk fate, respectively. Linking this knowledge with RUNX1 P'n, we discovered that the Mk bias induced by slowing MEP cell cycling requires RUNX1, and that slowing the MEP cell cycle increases pS-RUNX1. We are now assessing the kinases responsible for P'n of RUNX1. Upon completion of these studies, we will have elucidated how cell cycle rate affects pS-RUNX1 levels to promote Mk fate specification. Primary human megakaryocyte-erythroid progenitors (MEP) can undergo fate specification to become megakaryocytic (Mk) or erythroid (E) progenitors. Despite their importance in maintaining the blood system, their fate specification mechanism remains elusive. Based on our single cell RNA-seq data implying that RUNX1 may be a key regulator of differential gene expression in MEP fate specification, we first found that RUNX1 overexpression in MEP promotes Mk over E specification while RUNX1 inhibition causes the opposite. Because RUNX1 mRNA and protein levels did not differ between Mk vs. E, we tested whether post-translational modifications differ, and found that the level of phospho-S276 RUNX1 (pS-RUNX1) is significantly higher in Mk than E. We compared the effects on MEP fate specification of WT RUNX1 vs. RUNX1-4D (S249D, S266D, S276D and T303D mutations to mimic phosphorylation (P'n) at these sites) vs. RUNX1-4A (S249A, S266A, S276A, T303A to prevent P'n). RUNX1-4D promotes Mk specification more dramatically than WT or RUNX1-4A. To determine whether P'n of these S/T residues affects RUNX1 chromatin localization and gene expression patterns, we performed CUT&RUN of HA-tagged RUNX1 WT, 4A and 4D overexpressed in the HEL cell line, which models MEP, as well as RNA-seq of these cells. While the 3 forms of RUNX1 have similar chromatin association sites, many Mk-related genes were differentially expressed. We have published that enhancing and suppressing MEP cell cycle promotes E and Mk fate, respectively. Linking this knowledge with RUNX1 P'n, we discovered that the Mk bias induced by slowing MEP cell cycling requires RUNX1, and that slowing the MEP cell cycle increases pS-RUNX1. We are now assessing the kinases responsible for P'n of RUNX1. Upon completion of these studies, we will have elucidated how cell cycle rate affects pS-RUNX1 levels to promote Mk fate specification.

DMAG, a novel countermeasure for the treatment of thrombocytopenia

BackgroundThrombocytopenia is one of the most common hematological disease that can be life-threatening caused by bleeding complications. However, the treatment options for thrombocytopenia remain limited.MethodsIn this study, giemsa staining, phalloidin staining, immunofluorescence and flow cytometry were used to identify the effects of 3,3ʹ-di-O-methylellagic acid 4ʹ-glucoside (DMAG), a natural ellagic acid derived from Sanguisorba officinalis L. (SOL) on megakaryocyte differentiation in HEL cells. Then, thrombocytopenia mice model was constructed by X-ray irradiation to evaluate the therapeutic action of DMAG on thrombocytopenia. Furthermore, the effects of DMAG on platelet function were evaluated by tail bleeding time, platelet aggregation and platelet adhesion assays. Next, network pharmacology approaches were carried out to identify the targets of DMAG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to elucidate the underling mechanism of DMAG against thrombocytopenia. Finally, molecular docking simulation, molecular dynamics simulation and western blot analysis were used to explore the relationship between DAMG with its targets.ResultsDMAG significantly promoted megakaryocyte differentiation of HEL cells. DMAG administration accelerated platelet recovery and megakaryopoiesis, shortened tail bleeding time, strengthened platelet aggregation and adhesion in thrombocytopenia mice. Network pharmacology revealed that ITGA2B, ITGB3, VWF, PLEK, TLR2, BCL2, BCL2L1 and TNF were the core targets of DMAG. GO and KEGG pathway enrichment analyses suggested that the core targets of DMAG were enriched in PI3K–Akt signaling pathway, hematopoietic cell lineage, ECM-receptor interaction and platelet activation. Molecular docking simulation and molecular dynamics simulation further indicated that ITGA2B, ITGB3, PLEK and TLR2 displayed strong binding ability with DMAG. Finally, western blot analysis evidenced that DMAG up-regulated the expression of ITGA2B, ITGB3, VWF, p-Akt and PLEK.ConclusionDMAG plays a critical role in promoting megakaryocytes differentiation and platelets production and might be a promising medicine for the treatment of thrombocytopenia.Graphical

EloA promotes HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity

Megakaryocytes (MKs) are the unique non-pathological cells that undergo polyploidization in mammals. The polyploid formation is critical for understanding the MK biology, and transcriptional regulation is involved in the differentiation and maturation of MKs. However, little is known about the functions of transcriptional elongation factors in the MK polyploidization. In this study, we investigated the role of transcription elongation factor EloA in the polyploidy formation during the MK differentiation. We found that EloA was highly expressed in the erythroleukemia cell lines HEL and K562. Knockdown of EloA in HEL cell line was shown to impair the phorbol myristate acetate (PMA) induced polyploidization process, which was used extensively to model megakaryocytic differentiation. Selective over-expression of EloA mutants with Pol II elongation activity partially restored the polyploidization. RNA-sequencing revealed that knockdown of EloA decelerated the transcription of genes enriched in the ERK1/2 cascade pathway. The phosphorylation activity of ERK1/2 decreased upon the EloA inhibition, and the polyploidization process of HEL was hindered when ERK1/2 phosphorylation was inhibited by PD0325901 or SCH772984. This study evidenced a positive role of EloA in HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

RON Kinase Is a Novel Therapeutic Target for Philadelphia-Negative Myeloproliferative Neoplasms

The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineage compartments. Activation of JAK/STAT signaling is a major driver of all Ph-negative MPNs. During disease progression, MPN patients experience increased pro-inflammatory cytokine secretion, leading to remodeling of the bone marrow microenvironment and subsequent fibrosis. The JAK inhibitor ruxolitinib is an approved targeted therapy for MPN patients and has shown promise in its ability to reduce splenomegaly and the cytokine storm observed in patients. However, JAK inhibitors alone are not sufficient to reduce bone marrow fibrosis or to eliminate the JAK2-mutated clone. Furthermore, JAK inhibitor persistence, or reactivation of JAK/STAT signaling upon chronic JAK inhibitor treatment, has been observed in both MPN mouse models and MPN patients. Therefore, there is an urgent need for new treatment options in MPN. The tyrosine kinase RON, a member of the MET kinase family, has well-characterized roles in erythroblast proliferation and pro-inflammatory cytokine production. RON can be phosphorylated by JAK2 to stimulate erythroblast proliferation. However, the role of RON in MPN pathogenesis is unknown.We found that the ALK/MET/RON/ROS1 inhibitor crizotinib inhibited colony formation by MPN patient CD34+ cells, regardless of their disease subtype, mutation status, or JAK2 inhibitor treatment history (Figure 1A). To determine whether this is due to inhibition of the JAK/STAT signaling pathway, we performed phospho-flow cytometry of STAT3 and STAT5 in myelofibrosis patient erythroblasts treated with crizotinib ex vivo as well as Western blot analysis in the JAK2-mutated cell lines SET2 and HEL. We found that crizotinib inhibits the phosphorylation of JAK2, STAT3, and STAT5 (Figure 1B). Since crizotinib has not been reported to directly inhibit JAK2, we asked whether these effects of crizotinib in MPN cells could be explained by RON inhibition. Consistent with this hypothesis, we observed that shRNA knockdown of multiple RON isoforms also decreases the phosphorylation of JAK2, STAT5, and STAT3 in HEL cells (Figure 1C-D).To determine whether crizotinib can alter the MPN disease course in vivo, we tested crizotinib by oral gavage in the MPLW515L bone marrow transplant murine model of myelofibrosis at 100mg/kg daily for 2 weeks. We showed that crizotinib decreased the disease burden of MPL-W515L mice, as evidenced by decreased spleen and liver weights (Figure 1E). To determine the effects of RON genetic deletion on MPN pathogenesis, we tested whether genetic deletion of Stk (mouse gene for RON) impairs disease progression in the JAK2V617F bone marrow transplant MPN model by transplanting Stk-/- c-Kit+ bone marrow cells transduced with the JAK2V617F-GFP retrovirus into lethally irradiated recipients. We observed a significant delay in disease onset in Stk-/- transplant recipients compared to WT controls (Figure 1F). However, we found that Stk-/- mice have normal numbers of hematopoietic stem and progenitor cells, and normal bone marrow myeloid colony forming capacity, suggesting that RON is a safe therapeutic target.To determine whether RON plays a role in the JAK inhibitor persistence phenotype, we generated persistent cells by treating SET2 cells with increasing doses of ruxolitinib over 8 weeks, and confirmed persistent proliferation and JAK/STAT activation. Interestingly, we found that RON phosphorylation is enhanced in JAK inhibitor persistent cells, and that dual inhibition of RON and JAK2 overcomes JAK inhibitor persistence in SET2 cells (Figure 1G-H), suggesting that RON may potentiate the JAK2 persistence phenotype in response to ruxolitinib. Importantly, we showed by immunoprecipitation that phospho-RON and phospho-JAK2 physically interact in JAK inhibitor persistent SET2 cells, and that this interaction is disrupted by crizotinib (Figure 1I). In summary, our data demonstrate that RON kinase is a novel mediator of JAK/STAT signaling in MPNs, and that it plays a particularly important role in JAK inhibitor persistence. Our work suggests that therapeutic strategies to inhibit RON, such as crizotinib, should be investigated in MPN patients. [Display omitted] DisclosuresHalmos: Guardant Health: Membership on an entity's Board of Directors or advisory committees; Apollomics: Membership on an entity's Board of Directors or advisory committees; TPT: Membership on an entity's Board of Directors or advisory committees; Eli-Lilly: Research Funding; Advaxis: Research Funding; Blueprint: Research Funding; Elevation: Research Funding; Mirati: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Gritsman: iOnctura: Research Funding.

A Phase 2 Study to Evaluate the Efficacy and Safety of Selinexor in Patients with Myelofibrosis Refractory or Intolerant to JAK Inhibitors

▪Background:Selinexor is an oral, small molecule, selective inhibitor of nuclear export (SINE) compound that specifically blocks the karyopherin protein exportin 1 (XPO1, CRM1). In an shRNA library screen, we discovered that the survival of JAK2V167F mutant HEL cells is dependent on XPO1-mediated nuclear-cytoplasmic transport. Selinexor selectively suppressed primary myelofibrosis (MF) cells as compared with normal progenitor cells and induced hematologic responses in an MPN mouse model.Methods:An open label, prospective, investigator-initiated single center study is ongoing in adults with primary or secondary MF with resistance or intolerance to JAK inhibitor (JAKi) therapy with platelets > 30 K/μL and neutrophils > 500/μL. Selinexor was given orally once a week. Spleen volume was assessed by MRI at week 12 and week 24. The study was amended to include additional MRIs every 12 weeks in the year 1 and 24 weeks in the year 2. Primary end point is spleen response, defined as ≥ 35% spleen volume reduction (SVR) by MRI or CT, where applicable) at week 24. Bone marrow was evaluated at baseline and at week 24. The projected sample size of 24 will provide 83% power to reject a response rate of 15% and allow for up to a 25% dropout rate. We provide an interim report after completing 50% enrollment.Results:Between May 2019 and February 2021, 12 patients (pts) were enrolled. JAK2, CALR and MPL mutations were present in 7 (58.3%), 4 (33.3%) and 1 (8.3%) pts respectively. Eight pts (66.6%) had at least one high molecular risk mutation at baseline (Table 1). Median duration of prior JAKi therapy was 22 months (0.5 to 96 months) and 11 out of 12 were refractory to ruxolitinib at study enrollment. Median baseline spleen volume was 1454 cm 3(range 835 to 5792). Selinexor starting dose was 80 mg weekly in the first 6 pts and 60 mg for subsequent pts. At data cutoff, median duration of selinexor therapy was 36 weeks (range 11-114 weeks). One pt was not response evaluable and died due to liver abscess at week 12 (unrelated). One pt discontinued selinexor at week 18 due to grade 3 fatigue and was not evaluable for the primary end point. Of the 11 pts who had week 12 MRI or CT, 6 showed ≥ 10% SVR, 3 showed ≥ 25% SVR and 1 pt had early progression (Figure 1). At week 24, 5/9 (56%) pts had ≥ 25% SVR and 2/9 (22%) had ≥ 35% SVR (Figure 1). In 9 pts who had ≥24 weeks of selinexor, SVR ≥ 25% and ≥ 35% occurred at any point during study treatment in 4 (44%) and 3 (33%) pts, respectively. Two pts were red cell transfusion dependent at baseline; 1 became transfusion independent after 36 weeks of treatment, has not required transfusion for 49 weeks and remains on study treatment to date (114 weeks). Six pts (50%) discontinued selinexor. Reasons for treatment discontinuation are death in 1 pt, progressive disease in 1 pt, alternative treatment in 2 pts, and toxicity in 2 pts. Ten pts required dose reduction due to fatigue (1pt), anemia (1 pt), thrombocytopenia (2 pts), abdominal pain (1pt) and weight loss (5 pts). The most common treatment related adverse event was weight loss (grade 2 in 4 pts and grade 3 in 1 pt). This was manageable with treatment interruption and dose reduction, except in one pt who discontinued selinexor. As yet no changes in reticulin fibrosis MF grade were observed among 9 patients who received at least 24 weeks of treatment.Conclusions:Once weekly, oral selinexor showed single agent activity with sustained spleen responses in pts with JAKi refractory MF. Long-term administration of selinexor was well tolerated over time in MF pts. Correlatives studies including circulating inflammatory cytokine levels and mutant allele burden, as well as clonality studies by X-chromosome inactivation studies in woman, are underway and will be presented. [Display omitted] DisclosuresTantravahi: BMS: Research Funding; Novartis: Research Funding; CTI BioPharma: Research Funding; Abbvie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Consultancy, Honoraria, Research Funding. Patel: Stemline: Research Funding; Genentech: Research Funding; Roche: Research Funding. Chamoun: Karyopharm Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Shah: Karyopharm: Current Employment. George: Celgene: Consultancy; Bristol Meyers Squibb: Consultancy; Incyte Corporation: Consultancy; Blueprint Medicines: Consultancy. Deininger: Fusion Pharma, Medscape, DisperSol: Consultancy; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Novartis: Consultancy, Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding.

Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis.

Simple SummaryMyelofibrosis (MF) is a progressive myeloproliferative neoplasm with tendency towards leukemic transformation and has the poorest prognosis amongst the Philadelphia-negative classical myeloproliferative neoplasms (MPN). Ruxolitinib, the first FDA-approved JAK1/2 tyrosine kinase inhibitor, is efficient in reducing spleen size and improving patient symptoms, but it has not been shown to eradicate the MF clone. In this study, using functional genomics techniques, we identified the proteasome family as an additional therapeutic target and demonstrated that inhibition of the proteasome family by carfilzomib in combination with ruxolitinib enhanced suppression of primary MF cells in vitro.As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.

Active constituents of Zanthoxylum nitidium from Yunnan Province against leukaemia cells in vitro

Zanthoxylum nitidium (Roxb.) DC (Rutaceae) is well known for inhibiting the proliferation of human gastric, liver, kidney and lung cancer cells, though research on its potential use in treating leukaemia is relatively rare. Twenty-six compounds were isolated from the chloroform and petroleum ether extracts of the roots and leaves of Z. nitidium (Zanthoxylum nitidium). They were ( +)-9′-O-transferuloyl-5, 5′-dimethoxylaricriresinol (1), 8-(3′-oxobut-1′-en-1′-yl)-5, 7-dimethoxy-coumarin (2), 5, 7, 8-trimethoxy-coumarin (3), 5-(3′, 3′-dimethyl-2′-butenyloxy)-7, 8-dimethoxy-coumarin (4), 2-(5-methoxy-2-methyl-1H-indol-3-yl) methyl acetate (5), 2′-(5, 6-dihydrochleletrythrine-6-yl) ethyl acetate (6), 6-acetonyldi-hydrochelerythrine (7), 6β-hydroxymethyldihydronitidine (8), bocconoline (9), zanthoxyline (10), O-methylzanthoxyline (11), rhoifoline B (12), N-nornitidine (13), nitidine (14), chelerythrine (15), 4-hydroxyl-7,8-dimethoxy-furoquinoline (16), dictamnine (17), γ-fagarine (18), skimmianine (19), robustine (20), R-( +)-platydesmine (21), 4-methoxyl-1-methyl-2-quinoline (22), 4-methoxy-2-quinolone (23), liriodenine (24), aurantiamide acetate (25), 10-O-demethyl-12-O-methylarnottianamide (26). Four among them, compounds 4 – 6 and 16, were first confirmed in this study by UV, IR, 1D, 2D NMR and HR-ESI–MS spectra. Compounds 1 – 2 and 11 were isolated from Z. nitidium for the first time. Of the assayed compounds, 1, 2, 9, 10, 14, 15 and 24, exhibited good inhibitory activities in the leukaemia cell line HEL, whereas compound 14 (IC50: 3.59 µM) and compound 24 (IC50: 15.95 µM) exhibited potent inhibitory activities. So, to further investigate the possible mechanisms, cell cycle and apoptosis assays were performed, which indicated that compound 14 causes obvious S-phase arrest in HEL cells and induced apoptosis, whereas compound 24 only induced apoptosis. The present results suggested both compounds 14 and 24 are promising potential anti-leukaemia drug candidates.

A natural acylphloroglucinol triggered antiproliferative possessions in HEL cells by impeding STAT3 signaling and attenuating angiogenesis in transgenic zebrafish model

Leukemia is responsible for a reason of death, globally. Even though there are several treatment regimens available in the clinics against this disease, a perfect chemotherapeutic agent for the same is still under investigation. Natural plant-derived secondary metabolites are used in clinics to treat leukemia for better benefits with reduced side-effects. Likely, several bioactive compounds from Callistemon sp. were reported for their bioactive benefits. Furthermore, acylphloroglucinol derivatives from Callistemon salignus, showed both antimicrobial and cytotoxic activities in various adherent human cancer cell lines. Thus, in the present study, a natural acylphloroglucinol (2,6-dihydroxy-4-methoxyisobutyrophenone, L72) was tested for its antiproliferative efficacy in HEL cells. The MTT and the cell cycle analysis study revealed that L72 treatment can offer antiproliferative effects, both time and dose-dependent manner, causing G2/M cell cycle arrest. The western blot analysis revealed that L72 treatment triggered intrinsic apoptotic machinery and activated p21. Likewise, L72 could downregulate the gene expressions of XIAP, FLT3, IDH2, and SOD2, which was demonstrated by qPCR analysis, thus promoting its antiproliferative action. The L72 could impede STAT3 expression, which was evidenced by insilico autodock analysis and western blot analysis using STAT3 inhibitor, Pimozide. The treatment of transgenic (Flk-1+/egfr+) zebrafish embryos resulted in the STAT3 gene inhibition, proving its anti-angiogenic effect, as well. Thus, the study revealed that L72 could act as an antiproliferative agent, by triggering caspase-dependent intrinsic apoptosis, reducing cell proliferation by attenuating STAT3, and activating an anti-angiogenic pathway via Flk-1inhibition.

Broad spectrum anti-coronavirus activity of a series of anti-malaria quinoline analogues

In this study, a series of 10 quinoline analogues was evaluated for their in vitro antiviral activity against a panel of alpha- and beta-coronaviruses, including the severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2), as well as the human coronaviruses (HCoV) 229E and OC43. Chloroquine and hydroxychloroquine were the most potent with antiviral EC50 values in the range of 0.12–12 μM. Chloroquine displayed the most favorable selectivity index (i.e. ratio cytotoxic versus antiviral concentration), being 165 for HCoV-OC43 in HEL cells. Potent anti-coronavirus activity was also observed with amodiaquine, ferroquine and mefloquine, although this was associated with substantial cytotoxicity for mefloquine. Primaquine, quinidine, quinine and tafenoquine only blocked coronavirus replication at higher concentrations, while piperaquine completely lacked antiviral and cytotoxic effects.A time-of-addition experiment in HCoV-229E-infected HEL cells revealed that chloroquine interferes with viral entry at a post-attachment stage. Using confocal microscopy, no viral RNA synthesis could be detected upon treatment of SARS-CoV-2-infected cells with chloroquine. The inhibition of SARS-CoV-2 replication by chloroquine and hydroxychloroquine coincided with an inhibitory effect on the autophagy pathway as visualized by a dose-dependent increase in LC3-positive puncta. The latter effect was less pronounced or even absent with the other quinolines.In summary, we showed that several quinoline analogues, including chloroquine, hydroxychloroquine, amodiaquine, ferroquine and mefloquine, exhibit broad anti-coronavirus activity in vitro.

Abstract 1345: Pharmacodynamic biomarkers for Pim inhibition with TP-3654 in patients with solid tumors

Abstract Proviral integration site for Moloney murine leukemia virus-1 (Pim-1) is a serine/threonine kinase downstream of Jak/Stat signaling which promotes cell growth, survival, and drug resistance. Pim-1 kinase is an important driver of tumorigenesis and tumor survival through its role in a number of downstream pathways, including inhibition of apoptosis through phosphorylation of the BH3-only protein BAD. Pim-1 is expressed at very low levels in most normal tissues, but is overexpressed in many cancers, such as prostate, colorectal, and many hematologic malignancies. Pim-1 kinase activity is constitutive and therefore directly proportional to protein expression. As such, Pim-1 is an attractive therapeutic target. TP-3654 is a second-generation, oral Pim inhibitor currently in Phase I clinical trials in solid tumors and myelofibrosis (NCT03715504 and NCT04176198). TP-3654 inhibits all three Pim kinases, with Ki values for Pim-1 (5nM), and Pim-2 and Pim-3 &lt250 nM in a biochemical assay. Treatment with TP-3654 in Jurkat and HEL cell lines showed dose-dependent modulation of the downstream targets of PIM, such as pS6K and pBad. A dose-dependent increase of TP-3654 Cmax was observed in plasma and subcutaneous PC-3 xenograft tumors in animals dosed orally at 50, 100 and 200 mg/kg with Tmax at 1 hour. Animals bearing HEL tumors were dosed similarly and Cmax values increased in a dose-dependent manner with Tmax at 2 hours. Plasma and tumor TP-3654 concentrations were above the measurement threshold out to 24 hours post dose. We further explored pharmacodynamic biomarkers of Pim inhibition (pBad, pS6K, and pS6RP) in flash frozen (FF) tumor tissue at various time points in both models by western blot and saw a maximum of 40% decrease in phosphorylated S6K at 2 hours, and an 80% decrease in pBad at 8 hrs in PC3 xenograft tumors in mice treated with 100 mg/kg of TP-3654. We hypothesized that treatment with TP-3654 would modulate Pim signaling in both cancer cells and peripheral blood mononuclear cells (PBMCs). This allows for detection of pharmacodynamic markers using repeated peripheral blood draws instead of invasive biopsies. We treated PBMCs from multiple healthy human donors with TP-3654 ex vivo at 0.3 and 3 µM to assess the movement of Pim biomarkers. Two phosphorylation markers, pS6K and pS6RP consistently exhibited dose-dependent decreases (30-70%) as measured by western blot. Inhibition of pBad was dependent on pretreatment phosphorylation state. These results were confirmed in an automated western blot system. Citation Format: Curtis A. Allred, Yuta Matsumura, Ethika Tyagi, Mark Wade, Clifford Whatcott, Jason Foulks, Adam Siddiqui, David J. Bearss, Steven L. Warner. Pharmacodynamic biomarkers for Pim inhibition with TP-3654 in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1345.

Selective Inhibition of Nucleocytoplasmic Transport Overcomes Ruxolitinib Resistance in Myelofibrosis

Myelofibrosis (MF) is a stem cell-derived hematopoietic malignancy characterized by constitutive activation of JAK/STAT signaling through mutations in JAK2, calreticulin or MPL. MF patients have reduced life expectancy due to cytopenias, progression to acute myeloid leukemia and thromboembolic events. JAK kinase inhibitors such as ruxolitinib reduce constitutive symptoms but are not curative, with persistence of malignant cells. Allogeneic stem cell transplant remains the only curative therapy for MF, but is associated with considerable morbidity and mortality, defining an urgent need for new therapies that induce more profound and durable responses.We used a barcoded lentiviral shRNA library to identify survival-critical genes for MF in HEL cells (homozygous for JAK2V617F), established from a patient with erythroleukemia. shRNAs targeting RAN and binding protein (RANBP) were significantly depleted, implicating nucleocytoplasmic transport (NCT) as one of the top survival-critical pathways. RAN is a small GTPase, whose cycling between GTP- and GDP-bound forms provides the engine for NCT. We previously presented results showing that shRNA-mediated knockdown of RAN, significantly reduced proliferation and induced apoptosis in HEL and SET-2 cells (harboring heterozygous JAK2V617F). Additionally, KPT-330 (selinexor, Karyopharm Therapeutics), an inhibitor of CRM1, the core component of NCT, also inhibited proliferation of HEL and SET-2 cells and colony formation by primary MF CD34+ cells, but not cord blood (CB). Importantly, inhibition of NCT was effective at reducing colony formation in samples from ruxolitinib-refractory MF patients, regardless of mutation status. A clinical trial to evaluate KPT-330 in ruxolitinib-refractory MF is in preparation.KPT-8602 is a second-generation inhibitor of CRM-1. Unlike KPT-330, KPT-8602 does not cross the brain-blood barrier, resulting in minimal anorexia, malaise, and weight loss [Etchin et al., Leukemia, 2017 Jan;31(1):143-150]. To determine the efficacy of KPT-8602 in MF, we tested KPT-8602 in cell lines, patient samples, and a mouse model of MF, along with ruxolitinib as comparison.In cell proliferation assays, KPT-8602 was five-fold more potent than ruxolitinib against HEL cells (IC50: 83 nM vs. 437 nM) and as potent as ruxolitinib in SET-2 cells (IC50: 95 nM vs. 85 nM). In clonogenic assays, KPT-8602 showed selectivity toward primary MF vs. CB CD34+ cells (IC50: 25 nM for MF vs. 258 nM for CB) and, in combination with ruxolitinib, resulted in a 95% reduction in colony formation. KPT-8602 inhibited colony formation by CD34+ cells from all MF patients tested, including ruxolitinib-refractory cases, irrespective of mutation status (including triple negative).To evaluate the possible use of KPT-8602 in vivo, we used a mouse transduction/transplantation model of JAK2V617F - positive myeloproliferative neoplasm (MPN). In this model, mice develop MPN resembling polycythemia vera that progresses to MF [Bumm et al., Cancer Res., 2006, 1;66(23):11156-65]. MPN was evident based on elevated blood counts and blood GFP+ cells (representing JAK2V617F allelic burden). MPN mice were randomized into four groups: vehicles, KPT-8602 (initial dose 15 mg/kg daily, orally), ruxolitinib (initial dose 50 mg/kg twice daily, orally) or combined ruxolitinib with KPT-8602 at these same concentrations, and treated for 28 days. KPT-8602 as a single agent reduced red blood cells (RBC), white blood cells (WBC), BM GFP+ cells, peripheral blood (PB) GFP+ cells and spleen size to the same extent as ruxolitinib, and the combination was no more effective than either single agent (Table 1). Spleen cytoarchitecture was fully restored by KPT-8602, but bone marrow fibrosis was not rectified in any treatment group. Mice in all groups, including the combined vehicle controls, still experienced weight loss and lethality, suggesting that toxicity may be due to vehicles, and that optimization of drug formulations and dosing is warranted.Overall, these results indicate that NCT is critical for MF survival, and that NCT inhibition may provide therapeutic benefit for MF patients. KPT-8602 is being tested in a clinical trial (NCT02649790) for refractory multiple myeloma. The results of our study provide strong rationale for a clinical trial in patients with refractory MF. [Display omitted] DisclosuresBaloglu:Karyopharm Therapeutics Inc: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Celgene: Research Funding; ARIAD: Consultancy; Gilead: Research Funding; BMS: Consultancy, Research Funding.

The Pharmacological IGF1R-IRS1/2 Inhibitor NT157 Presents Multiple Anti-Neoplastic Effects in Myeloproliferative Neoplasms

Introduction: The recurrent V617F mutation in JAK2 plays an important role in the pathogenesis of BCR-ABL1 negative myeloproliferative neoplasms (MPN). Absence of complete clinical response to ruxolitinib, a JAK1/2 inhibitor, highlights the need to target other signaling pathways that contribute to JAK2. Recent evidence indicates that the IGF1R/IRS pathway is a potential target in MPN: (i) insulin receptor substrate 2 (IRS2) cooperates with malignant transformation induced by JAK2V617F, and (ii) increased IGF1 signaling induced by AIRAPL down-regulation causes MPN in mice. NT157 is a synthetic compound designed as an IRS1/2 inhibitor, which presented anti-neoplastic effects in solid tumors. Aims: The objective herein was tocharacterize the molecular and cellular effects of NT157 treatment, combined or not with ruxolitinib, in MPN JAK2V617F. Methods: HEL and SET2 cell lines (both carrying JAK2V617F mutation) were treated or not with vehicle, NT157 and/or ruxolitinib and submitted to evaluation of cell viability (MTT assay), apoptosis (annexin V/PI and caspase cleavage), proliferation (Ki-67), clonogenicity (colony forming assay), cell cycle (PI), gene expression (PCR array and qPCR) and protein expression/activation (Western blot). Primary cells from polycythemia vera (PV) patients (n=3) were exposed to NT157 (3.2 µM) treatment and evaluated for erythropoietin-independent colony formation. Statistical analysis was performed using ANOVA or Student's t test. P -value <0.05 was considered statistically significant. Results: NT157 treatment significantly decreased cell viability, clonogenicity and cell proliferation (all p <0.05). NT157 induced apoptosis at doses equal or higher than 0.8 μM as verified by flow cytometry analysis of annexin V/PI (p ˂0.001) and confirmed by increased levels of cleaved caspase 3, 8 and 9. The percentage of apoptotic cells (annexin V positive) identified for untreated cells and cells exposed to NT157 0.8 μM was respectively, 14 and 56% after 24 hours of treatment; 11 and 60% after 48 hours, 6 and 40% after 72 hours (all p ˂0.001). Cell cycle analysis was performed 24 hours after NT157 exposition and revealed cell cycle arrest in G2/M, associated with decrease in G0/G1 population (p ˂0.001). In a molecular scenario, NT157 treatment strongly reduced IRS1 and IRS2 expression, and STAT3, STAT5 and ERK1/2 phosphorylation in a dose dependent manner. PCR array analyzes revealed that NT157 treatment modulated 23 oncogenes and tumor suppressor genes. Based on the cellular effects observed after NT157 treatment, we selected six genes, three repressed oncogenes and three upregulated apoptotic-related genes, to be validated in a larger number of experiments. Upon NT157 exposition, CCND1 (cyclin D1), MYB and WT1 mRNA levels were reduced, while CDKN1A (p21), FOS and JUN mRNA levels increased (all p ˂0.05). The combined treatment of NT157 plus ruxolitinib did not present potentializing effects when compared to the monotherapies. In contrast, the combined treatment prevented, at least in part, the cytotoxic effects of NT157. In HEL cells, NT157 0.8 µM monotherapy promoted enhanced apoptosis rate compared to the combined treatment, using NT157 0.8 plus ruxolitinib 0.3 µM (52% vs. 38%, respectively [ p ˂0.05]). Ruxolitinib also prevented NT157 effect on cell cycle progression, causing a reduction in G2/M arrest when compared to NT157 monotherapy (p <0.05). In primary hematopoietic cells from PV patients, NT157 exposition reduced erythropoietin-independent colony formation by 49 % (patient #1), 12% (patient #2) and 23% (patient #3); the mean reduction of erythroid colony number for all PV samples was 29% (p <0.05). Conclusions: IGF1R-IRS1/2 pharmacological inhibitor NT157 displayed remarkable anti-neoplastic effects in JAK2V617F cells lines and MPN primary cells. Our exploratory approaches provided new insights about molecular mechanisms of NT157. Our findings indicate that inhibition of IGF1R/IRS signaling, using NT157 monotherapy, emerges as a novel therapeutic opportunity for MPN JAK2V617F. DisclosuresNo relevant conflicts of interest to declare.

Human Germline HLTF E259K Mutation Identified in Familial MDS Patients Accumulates DNA Damage through Impaired PCNA Polyubiquitination

Although several causal genes of familial myelodysplastic syndromes (MDS) have been discovered, the genetic landscape and the molecular pathogenesis still remain unclear. To explore novel causal genetic alterations and their pathogenetic significance, we performed whole-exome sequencing analysis using four samples from a Japanese familial MDS pedigree, two affected and two unaffected as controls. In total, 16889 non-synonymous germline substitutions were obtained. Among them, twelve candidate single-nucleotide variations (ANK1, C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, RYK, TTN) were elucidated. Of the candidates, we elucidated HLTF, encoding a member of the SWItch/Sucrose Non-Fermenting (SWI/SNF) family, as a putative associated gene. HLTF works as an E3-ubiquitin ligase and binds with Methyl Methanesulfonate Sensitive 2 (MMS2)/Ubiquitin Conjugating Enzyme E2N (UBC13) complex. This complex is known to promote the Lys-63 linked polyubiquitination of proliferating cell nuclear antigen (PCNA), followed by an error-free postreplication DNA repair by arranging template switching (Mailand et al. Nat Rev Mol Cell Biol 2013). The obtained germline mutation of HLTF was p.Glu259Lys (E259K), heterozygous mutation.To study the functional role of HLTF, we firstly evaluated the effects of Hltf silencing on hematopoiesis by in vitro colony-formation assay with Hltf-knocked down mouse primary bone marrow cells, and found that these cells have enhanced in vitro colony-formation capacity. In agreement with the acquisition of an immature phenotype, the cells expressing c-kit, a hematopoietic stem/ progenitor cell marker, were increased in Hltf shRNA-expressing colonies after the first round. We then focused on DNA damage in HLTF knockdown HEL cells according to the function of HLTF post-replication DNA repair. We examined the phosphorylation of gamma H2AX foci by immunofluorescent staining and found that the number of foci was increased in HLTF knockdown cells compared with control cells, suggesting that DNA damage was accumulated in HLTF knockdown cells.To investigate the effects of exogenous expression of HLTF E259K mutant protein, we evaluated in vitro apoptosis status with HEL cells transduced with empty vector (EV), HLTF wild type (WT), and HLTF E259K mutant (MT), respectively. In apoptosis assay, annexin V positive cells were significantly increased in MT-overexpressed HEL cells compared with WT-overexpressed HEL cells. In addition, we found that the number of gamma H2AX foci was increased in HLTF MT-overexpressed HEL cells, suggesting that DNA double-strand breaks were accumulated in MT-overexpressed cells. To explore the mechanism of accumulated DNA double-strand breaks by mutant HLTF, we evaluated the binding ability of HLTF mutant protein with PCNA, and MMS2/UBC13, all of which play a critical role in the DNA double-strand break repair process. We found that binding of HLTF MT to MMS2 or UBC13 was significantly attenuated compared with HLTF WT although binding to PCNA remained unchanged, suggesting HLTF MT cannot efficiently form a complex that induces polyubiquitination of PCNA. In line with this, PCNA polyubiquitination was impaired in HLTF MT-overexpressed cells compared with HLTF WT-overexpressed cells.Finally, we searched somatic HLTF mutation in sporadic MDS bone marrow samples. Whole HLTF coding regions of 40 samples were sequenced. As a result, a novel HLTF missense heterozygous mutation (p.Thr50Ala, T50A) was identified in one sample (1/40 = 2.5%). These data suggested the possibility that HLTF mutation might contribute to the pathogenesis not only in familial MDS but also in sporadic MDS.In conclusion, our results indicated that HLTF E259K germ line mutation accumulates DNA damage through impaired PCNA polyubiquitination. The aberrant function of HLTF mutant might play a role in the pathogenesis of familial MDS. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Suzuki:Bristol-Myers K.K: Honoraria; Novarltis: Honoraria; Celgen: Honoraria; Ono: Honoraria; Fujimoto: Honoraria; Takeda: Honoraria. Harada:NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding; Celgene: Research Funding. Kurokawa:Bristol-Myers Squibb: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Pfizer Inc.: Research Funding; Celgene Corporation: Speakers Bureau.

Critical Interferon-Response Genes Are Induced By JAK2V617F but Repressed By BCR-ABL, and They Are Responsible for the Differential Effects of Interferon in PV Vs. CML

Interferon-alpha (IFNa) is successfully used as monotherapy in JAK2V617F positive myeloproliferative neoplasms (MPN) such as polycythemia vera (PV), but with only limited success in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa in JAK2V617F vs. BCR-ABL positive cells.In 32D cells stably transduced with JAK2V617F, IFNa alone (100 U/mL) significantly decreased the viability to less than 50% of control but had no significant effect on 32D-BCR-ABL cell viability, as assessed by MTT assays. Gene expression microarray data of 32D-JAK2V617F cells revealed strong upregulation of a number of interferon-stimulated genes compared to empty vector-transduced (EV) cells, and RT-PCR validation confirmed significantly increased expression of Stat1, Stat2 and Irf9 and reversal of these effects by ruxolitinib treatment. Conversely, BCR-ABL led to a decrease of these three genes, and this was reversed by imatinib. Similar responses were seen in human HEL and K562 cells. Histone (H3K9) acetylation of Stat1, Stat2 and Irf9 promoters was significantly higher in 32D-JAK2V617F vs. 32-BCR-ABL cells, correlating with gene expression. IFNa treatment increased Stat1, Stat2 and Irf9 mRNA in both 32D-JAK2V617F and 32D-BCR-ABL cells, and this effect was reduced by ruxolitinib but potentiated by imatinib. In line with these results, the phosphorylation of STAT1 (P-STAT1) was induced by IFNa in all cell lines. In addition, Stat1, Stat2 and Irf9 mRNA was significantly downregulated in peripheral blood samples from CML but not PV patients, as compared to healthy controls (all subjects provided written informed consent, as approved by the local ethics committee). Likewise, Stat1 and Stat2 expression was significantly downregulated in SCLtTA/BCR-ABL but not JAK2V617F transgenic mice.We next analyzed whether ectopic expression of STAT1 or STAT2 or their dominant-negative Y/F mutants affected the IFNa sensitivity of 32D-JAK2V617F vs. 32D-BCR-ABL cells. While there was little apoptosis in empty-vector- or STAT1-overexpressing 32D-BCR-ABL cells, overexpression of the STAT1Y701F mutant in these cells showed a significantly increased basal level of apoptosis, which was further enhanced by IFNa (p<0.05). This was associated with IFNa-induced P-STAT1 but complete absence of P-STAT2. Conversely, EV- or STAT1-overexpressing 32D-JAK2V617F cells showed strong IFNa-induced apoptosis, while STAT1Y701F overexpression in these cells led to abrogation of IFNa-induced apoptosis. This was associated with decreased P-STAT1 but preserved P-STAT2. On the other hand, STAT2 overexpression decreased P-STAT1 and P-STAT2 and led to mild IFNa-induced apoptosis in 32D-BCR-ABL cells, but preserved the high P-STAT1 and P-STAT2 as well as potent IFNa responsiveness in 32D-JAK2V617F cells.In conclusion, JAK2V617F and BCR-ABL induce opposite effects on IFNa target genes in vitro and in vivo, suggesting that JAK2V617F but not BCR-ABL sensitizes cells to interferon. Moreover, while the major factor of IFNa-induced apoptosis in JAK2V617F positive cells is an increase of P-STAT1, the major determinant in BCR-ABL positive cells is a decrease of P-STAT2. Together, these results may possibly explain the differences of clinical responses to IFNa monotherapy in PV vs. CML. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Koschmieder:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche: Other: Clinical Trial participation.

Effect of rhTPO to the Proliferation and Apoptosis of Acute Myeloid Leukemia Cell Lines

To investigate the effects of recombinant human thrombopoietin (rhTPO) to proliferation and apoptosis of acute myeloid leukemia (AML) cell lines. After the treatment of different concentrations of rhTPO (0, 50, 100 ng/ml) for different time (24,48,72 h)，the cell proliferation rates of the AML cell lines (Kasumi-1, Skno-1, HEL, HL-60, THP-1) were determined by CCK-8 method. Apoptosis rate of each cell line cocultured with rhTPO was detected by Annexin V/PI method. The relative expression of TPO receptor c-MPL (myeloproliferative clonal antibody) mRNA in AML cell lines was detected by Q-PCR. The expression of c-MPL protein in each cell line was detected by Western blot. The expression of c-MPL antigen in HL-60 cells treated by different concentrations of rhTPO was detected by Flow cytometry. RhTPO showed no promotion to the proliferation of Kasumi-1, Skno-1, HEL, HL-60, THP-1 cell lines，however，it showed inhibitory effect to cell proliferation (72 h 0 ng/ml vs 100 ng/ml, P= 0.029) and pro-apoptotic (48 h 0 ng/ml vs 50 ng/ml, P=0.0143) in HL-60 cells. In Kasumi-1, Skno-1, HEL and THP-1 cells, there showed no statistically significant differences in apoptosis rate among each groups treated by different concentrations of rhTPO. Each AML cell line showed different levels of c-MPL gene and c-MPL protein expression, but HEL cells showed the highest expression in both of them. After HL-60 cells were treated by different concentrations of rhTPO for 48 hours, there showed no statistical difference in c-MPL antigen expression among each groups. RhTPO can not promote the proliferation of Kasumi-1, Skno-1, HEL, HL-60 and THP-1 leukemia cell lines. On the contrary, rhTPO can inhibit HL-60 cell proliferation and promote its apoptosis, and this effect is not related to c-MPL gene expression or protein expression.

Nifuroxazide as JAK2 inhibitor: A binding mode proposal and Hel cell proliferation assay

Nifuroxazide has been employed as an anti-diarrheic agent since 1966, but in the last decade has brought to the research spotlight again due to its recently described antitumoral activity through the JAK2 inhibitory potential. Since 2008, more than 70 papers have been published about the issue and more are expected to the following years. Herein we discuss the findings of molecular modelling studies which were performed to elucidate the potential binding mode of this drug into the JAK2 ATP recognition site and also into the allosteric region near the catalytic site. Molecular modelling followed by dynamics simulations indicated the NFZ could bind at both sites, such as a Type II kinase inhibitor since residues from both ATP and modulatory site would exhibit contacts with the drug when in a stable complex. Synthesis of NFZ and its sulfur bioisosteric analogue GPQF-63 were performed and experimental assays against HEL cells indicate the potential of NFZ and, mainly of its analogue GPQF-63 in acting as inhibitors of cell growth. HEL-cells present the JAK2 V617F mutation which leads to an enhanced JAK/STAT pathway and they have never been tested by the NFZ activity before. A mechanistic approach was also performed and revealed that both compounds induce cell apoptosis.Taken together, both the theoretical and experimental approaches point out the N-acylhydrazones as good starting points in the search for JAK2 modulatory small molecules which could then, be studied as promising leads toward new alternatives to control the JAK-STAT pathway related pathologies. This is the first study, as far as we have known, to propose a potential binding mode for NFZ as well as reporting the activity of this drug against HEL cells, which are a usual cellular model to human erythroleukemia and other myeloproliferative diseases.

Deficiency of ARHGAP21 alters megakaryocytic cell lineage responses and enhances platelet hemostatic function

BackgroundMicrotubules, actin and Rho GTPase proteins are essential players in the megakaryocyte biology for platelet formation and function. ObjectivesTo investigate the role of ARHGAP21, a RhoGAP protein, in megakaryocytic differentiation and platelet function. MethodsCytoskeletal proteins were investigated in HEL cells silenced for ARHGAP21 and submitted to megakaryocyte differentiation. The role of Arhgap21 in platelet function was accessed using haploinsufficient (Arhgap21+/−) mice. Arhgap21+/− platelet aggregation and p-selectin exposure were evaluated in response to thrombin. Vessel occlusion time and thrombus formation were detected after injury of the carotid artery. Platelet morphology was accessed by electronic microscopy. ResultsARHGAP21 was upregulated during megakaryocytic differentiation of the cell line and primary mouse cells. In the HEL cell model, ARHGAP21 was detected in the cytoplasmic protrusions, colocalized and associated with α-tubulin and was mostly detected in the protein cell fraction containing the polymerized tubulin. Silencing of ARHGAP21 decreased the expression of Glu-tubulin, suggesting microtubule instability, and enhanced cell spreading. Platelets from Arhgap21+/− mice presented enhanced thrombin-induced aggregation and p-selectin exposure associated with increased size of α-granules. Arhgap21+/− mice also showed increased CDC42 and RHOA activities, shorter tail-bleeding and accelerated thrombus formation. ConclusionsOur results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement.

Mitochondria in human acute myeloid leukemia cell lines have ultrastructural alterations linked to deregulation of their respiratory profiles

Mitochondria not only are essential for cell metabolism and energy supply but are also engaged in calcium homeostasis and reactive oxygen species generation and play a key role in apoptosis. As a consequence, functional mitochondrial disorders are involved in many human cancers including acute myeloid leukemia (AML). However, very few data are available on the deregulation of their number and/or shape in leukemic cells, despite the evident link between ultrastructure and function. In this context, we analyzed the ultrastructural mitochondrial parameters (number per cell, mitochondria area, number of cristae/mitochondria, cristal thickness) in five leukemia cell lines (HEL, HL60, K562, KG1, and OCI-AML3) together with the functional assay of their respiratory profile. First, we describe significant differences in basal respiration, maximal respiration, ATP production, and spare respiratory capacity between our cell lines, confirming the various respiratory profiles among leukemia subtypes. Second, we highlight that these variations are obviously associated with significant interleukemia heterogeneity of the number and/or shape of mitochondria. For instance, KG1, characterized by the smallest number of mitochondria together with reduced cristal diameter, had a particularly deficient respiratory profile. In comparison, the HEL and K562 cell lines, both with high respiratory profiles, harbored the largest number of mitochondria/cells with high cristal diameters. Moreover, we report that K562, carrying the ASXL1 mutation, presents significant mitochondria-endoplasmic reticulum deficiency reflected by decreases in the numbers of matrix granules and mitochondria-associated endoplasmic reticulum membrane (MAM) and mitochondrial-derived vesicle (MDV) precursors, which are implicated in the regulatory pathways of cell mortality via the processes of mitophagy and calcium homeostasis. Contrarily, HL60 carried high levels of matrix granules and MAMs and had a higher sensitivity to drugs targeting mitochondria (rotenone/antimycin). We confirm the implication of ASXL1 mutation in this mitochondria dysregulation through the study of transcript expression (from 415 patients with public data) involved in three mitochondrial pathways: (1) endoplasmic reticulum-mitochondria contacts (MAMs), (2) matrix granule homeostasis, and (3) MDV precursor production. Our study offers new and original data on mitochondria structural alterations linked to deregulation of respiration profiles in AMLs and some genetic characteristics, suggesting that modifications of mitochondrial shape and/or number in leukemic cells participate in chemoresistance and could be a targeted mechanism to regulate their proliferative potential.

Rational drug design of benzothiazole-based derivatives as potent signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitors.

The cumulative evidence supports STAT3, a transcriptional mediator of oncogenic signaling, as a therapeutic target in cancer. The development of STAT3 inhibitors remain an active area of research as no inhibitors have yet to be approved for cancer treatment. In a continuing effort to develop more potent STAT3 inhibitors based on our previously identified hit compound 16w, a series of benzothiazole derivatives with unique binding mode in SH2 domain of STAT3 were designed, synthesized and biologically evaluated. Of note, compound B19 demonstrated excellent activity against IL-6/STAT3 signaling pathway with the IC50 value as low as 0.067μM as determined by a luciferase reporter assay. Moreover, multiple compounds displayed potent antiproliferative activity against MDA-MB-468 and JAK2 mutant HEL cell lines. Further biochemical study using Western blot assay indicated that B19 blocked the phosphorylation of STAT3 at Tyr 705 and Ser 727 and thus suppressed STAT3-mediated gene expression of c-MYC and MCL-1. Simultaneously, it induced cancer cell G2/M phase arrest and apoptosis both in MDA-MB-468 and HEL cell lines. Finally, molecular docking study along with surface plasmon resonance (SPR) and fluorescence polarization (FP) assays disclosed the binding mode of B19 in STAT3 SH2 domain. Taken together, our finding suggests that B19 is a promising therapeutic STAT3 inhibitor for cancer treatment.