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Platelets are essential components in the tumor microenvironment. For decades, clinical data have demonstrated that cancer patients have a high risk of thrombosis that is associated with adverse prognosis and decreased survival, indicating the involvement of platelets in cancer progression. Increasing evidence confirms that cancer cells are able to induce production and activation of platelets. Once activated, platelets serve as allies of cancer cells in tumor growth and metastasis. They can protect circulating tumor cells (CTCs) against the immune system and detachment-induced apoptosis while facilitating angiogenesis and tumor cell adhesion and invasion. Therefore, antiplatelet agents and platelet-based therapies should be developed for cancer treatment. Here, we discuss the mechanisms underlying the bidirectional cancer-platelet crosstalk and platelet-based therapeutic approaches.

Receptor tyrosine kinase ROR1 plays an essential role in embryogenesis and is overexpressed in many types of malignant tumors. Studies have demonstrated that it plays an important role in oncogenesis by activating cell survival signaling events, particularly the non-canonical WNT signaling pathway. Antibody-based immunotherapies targeting ROR1 have been developed and evaluated in preclinical and clinical studies with promising outcomes. However, small molecule inhibitors targeting ROR1 are underappreciated because of the initial characterization of ROR1 as a peusdokinase. The function of ROR1 as a tyrosine kinase remains poorly understood, although accumulating evidence have demonstrated its intrinsic tyrosine kinase activity. In this review, we analyzed the structural and functional features of ROR1 and discussed therapeutic strategies targeting this kinase.

It was previously published that single-nucleotide polymorphism rs2476601 (PTPN22 [protein tyrosine phosphatase non-receptor type 22]-C1858T) might be related to increased sensibility to Mycobacterium tuberculosis and M. leprae infection. However, the results were inconclusive despite a high degree of similarity between both parameters. Herein, we carried out this meta-analysis to systematically summarize and articulate the correlation between PTPN22-C1858T polymorphism and mycobacterial infection. The susceptibility of PTPN22-C1858T carriers with autoimmune conditions receiving immunosuppressive therapy to M. tuberculosis and M. leprae infection was determined. A systematic retrieval of studies on relevance of PTPN22-C1858T polymorphism to susceptibility of M. tuberculosis or M. leprae infection was performed in Chinese National Knowledge Infrastructure, PubMed and Embase databases. We regarded Odds ratios (ORs) and 95% confidence intervals (CIs) as the determined effect size. Finally, four and two case-control studies on tuberculosis and leprosy, respectively, were included. In all genetic models, without indicated association between PTPN22-C1858T polymorphism and tuberculosis’s susceptibility. [C versus T: OR = 0.22 (95% CI: 0.09–0.50, PH = 0.887); CT versus CC: OR = 0.21 (95% CI: 0.09–0.49, PH = 0.889); TT+CT versus CC: OR = 0.21 (95% CI: 0.09–0.49, PH = 0.889)]. A significantly increased risk of leprosy was perceived in patients with the PTPN22-C1858T polymorphism [C versus T: OR = 2.82 (95% CI: 1.02–7.81, PH = 0.108)]. While the PTPN22-C1858T polymorphism is irrelevant to higher susceptibility to the infection of M. tuberculosis in Caucasians and Asians, it is relevant to increased susceptibility to the infection of M. leprae. However, the results of M. leprae are supposed to interpreted with prudence owing to the limited quantity of studies and heterogeneity. Further well-designed studies with sufficient populations are required to verify our conclusions.

Background: Fms-like tyrosine kinase 3 (FLT3) gene mutations occur in approximately 30% of all AML patients. Internal tandem duplication (ITD) in the juxtamembrane domain and point mutations within the tyrosine kinase domain (TKD) are two distinct types of FLT3 mutations. FLT3-ITD has been determined as an independent poor prognostic factor, but the prognostic impact of FLT3-TKD remains controversial. Hence, we performed a meta-analysis to investigate the prognostic significance of FLT3-TKD in patients with AML. Methods: A systematic retrieval of studies on FLT3-TKD in patients with AML was performed in PubMed, Embase, and CNKI databases on Sept. 30th, 2020. Hazard ratio (HR) and its 95% confidence intervals (95% CIs) were used to determine the effect size. Meta-regression model and subgroup analysis were used for heterogeneity analysis. Begg’s and Egger’s tests were performed to detect potential publication bias. The sensitivity analysis was performed to evaluate the stability of findings in meta-analysis. Findings: Twenty prospective cohort studies (n=10970) on the prognostic effect of FLT3-TKD in AML were included: 9744 subjects with FLT3-WT and 1226 subjects with FLT3-TKD. We found that FLT3-TKD revealed no significant effect on DFS (HR=1.12, 95% CI: 0.90-1.41) and OS (HR=0.98, 95% CI: 0.76-1.27) in general. However, meta-regressions demonstrated that ethnicity contributed to the high heterogeneity observed in the prognosis of FLT3-TKD in AML. To be specific, FLT3-TKD represented a beneficial prognosis of DFS (HR=0.56, 95% CI: 0.37-0.85) and OS (HR=0.63, 95% CI: 0.42-0.95) for Asians while it represented an adverse prognosis of DFS for Caucasians with AML (HR=1.34, 95% CI: 1.07-1.67). Interpretation: Our results showed that FLT3-TKD revealed no significant effects on DFS and OS of patients with AML, which is consistent with the controversial status nowadays. Distinct prognosis of FLT3-TKD in AML between Asians and Caucasians may attribute to different chemotherapy regimens (including the dose of cytarabine and daunorubicin in conventional "3+7" induction and the clinical usage of HHT) and diverse genetic background. The dose of cytarabine (100 mg/m2) and daunorubicin (50 mg/m2) used in Asian countries in "3+7" induction therapy may benefit Caucasians with AML carrying FLT3-TKD. Funding Information: National Natural Science Foundation of China (NSFC, Grant No. 82000150). Declaration of Interests: We declare no competing interests.

Macrophages play a crucial role in host innate immune defense against infection and tissue injury. Macrophages are highly plastic cells and their subtypes have been characterized as M1 (also termed classically activated) and M2 (alternatively activated). Although the M1/M2 paradigm has been well documented, less is known regarding the role of macrophage activation/polarization in inflammation-associated necrotic cell death. To address this gap in current knowledge, we prepared bone marrow-derived macrophages, induced them to M1 or M2 subtypes, and then investigated the expression of necroptosis signaling molecules and macrophage subtype-dependent responses to different necroptosis inducers. We found that necroptosis effector mixed lineage kinase domain-like protein (MLKL) and the key necroptosis regulator Z-DNA/RNA binding protein 1 were predominantly induced in M1 but not M2 macrophages. Interestingly, the protein but not mRNA levels of receptor-interacting protein kinase-3 (RIPK3) were also upregulated in M1 macrophages. We further found that macrophage necrotic cell death, the releases of lactate dehydrogenase and dead cell proteases as well as MLKL phosphorylation at Ser345 in response to various necroptosis inducers were greatly augmented in M1 but not M2 macrophages, and the accelerated effects were blocked by two structurally distinct specific RIPK3 inhibitors GSK872 or GSK843. Thus, our findings demonstrate that M1 but not M2 subtypes of macrophages are more susceptible to inflammation-related lytic cell death in an RIPK3 kinase activity-dependent manner.

Background Acute myeloid leukemia (AML) is a malignant hematological neoplasm of myeloid progenitor cells. Mutations of FLT3 in its tyrosine kinase domain (FLT3-TKD) are found in ~ 8% of patients with AML, with D835Y as the most common substitution. This mutation activates survival signals that drives the disease and is resistant to the first generation FLT3 inhibitors. Development of a highly sensitive method to detect FLT3D835Y is important to direct therapeutic options, predict prognosis, and monitor minimal residual disease in patients with AML. Methods and results In the present study, we developed a highly sensitive FLT3D835Y detection method by using the restriction fragment nested allele-specific PCR technique. The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the FLT3D835Y mutation site, 2) digestion of the PCR products with restriction enzyme EcoRV that only cleaves the wild type allele, and 3) detection of FLT3D835Y by allele-specific PCR with nested primers. We were able to detect FLT3D835Y with a sensitivity of 0.001% by using purified plasmid DNAs and blood cell DNAs containing known proportions of FLT3D835Y. We analyzed blood cell DNA samples from 64 patients with AML and found six FLT3D835Y-positive cases, two of which could not be detected by conventional DNA sequencing methods. Importantly, the method was able to detect FLT3D835Y in a sample collected from a relapsed patient while the patient was in complete remission with negative MRD determined by flow cytometry. Therefore, our RFN-AS-PCR detected MRD after treatment that was missed by flow cytometry and Sanger DNA sequencing, by conventional methods. Conclusions We have developed a simple and highly sensitive method that will allow for detection of FLT3D835Y at a very low level. This method may have major clinical implications for treatment of AML.

The myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis and primary myelofibrosis are hematopoietic stem cell disorders and share driver mutations that either directly activate the thrombopoietin receptor, MPL, or activate it indirectly through gain-of-function mutations in the gene for JAK2, its cognate tyrosine kinase. Paradoxically, MPL surface expression in hematopoietic stem cells is also reduced in the myeloproliferative neoplasms due to abnormal post-translational glycosylation and premature destruction of JAK2, suggesting that the myeloproliferative neoplasms are disorders of MPL processing since MPL is the only hematopoietic growth factor receptor in hematopoietic stem cells. To examine this possibility, we genetically manipulated MPL expression and maturation in a JAK2V617F transgenic mouse model of polycythemia vera. Elimination of MPL expression completely abrogated the polycythemia vera phenotype in this JAK2V617F transgenic mouse model, which could only be partially restored by expression of one MPL allele. Most importantly, elimination of thrombopoietin gene expression abrogated the polycythemia vera phenotype in this JAK2V617F transgenic mouse model, which could be completely restored by expression of a single thrombopoietin allele. These data indicate that polycythemia vera is in part a thrombopoietin-dependent disorder and that targeting the MPL-thrombopoietin axis could be an effective, nonmyelotoxic therapeutic strategy in this disorder.

Background Myeloid leukemia represents a heterogeneous group of cancers of blood and bone marrow which arise from clonal expansion of hematopoietic myeloid lineage cells. Acute myeloid leukemia (AML) has traditionally been treated with multi-agent chemotherapy, but conventional therapies have not improved the long-term survival for decades. Chronic myeloid leukemia (CML) is an indolent disease which requires lifelong treatment, is associated with significant side effects, and carries a risk of progression to potentially lethal blast crises. Recent findings Recent advances in molecular biology, virology, and immunology have enabled researchers to grow and modify T lymphocytes ex-vivo. Chimeric antigen receptor (CAR) T-cell therapy has been shown to specifically target cells of lymphoid lineage and induce remission in acute lymphoblastic leukemia (ALL) patients. While the success of CAR T-cells against ALL is considered a defining moment in modern oncology, similar efficacy against myeloid leukemia cells remains elusive. Over the past 10 years, numerous CAR T-cells have been developed that can target novel myeloid antigens, and many clinical trials are finally starting to yield encouraging results. In this review, we present the recent advances in this field and discuss strategies for future development of myeloid targeting CAR T-cell therapy. Conclusions The field of CAR T-cell therapy has rapidly evolved over the past few years. It represents a radically new approach towards cancers, and with continued refinement it may become a viable therapeutic option for patients of acute and chronic myeloid leukemia.

Presenilin-associated protein (PSAP) was originally identified as a mitochondrial proapoptotic protein. To further explore the apoptotic pathway that involves PSAP, our yeast two-hybrid screen revealed that PSAP interacts with a death receptor, DR6. DR6 is a relatively less common member of the death receptor family and has been shown to mediate the neurotoxicity of amyloid-β, mutant SOD1, and prion proteins and has also been implicated in the regulation of immune cell proliferation and differentiation. Our previous study showed that DR6 induces apoptosis via a unique mitochondria-dependent pathway different from the conventional death receptor-mediated extrinsic apoptotic pathways. Thus, the interaction of DR6 with PSAP established a direct molecular link between DR6 and mitochondrial apoptotic pathway. We investigated the possible role of PSAP in DR6-induced apoptosis. Interestingly, it was discovered that knockdown of PSAP strongly inhibited DR6-induced apoptosis. To further elucidate the mechanism by which PSAP mediates DR6-induced mitochondria-dependent apoptosis, our data demonstrated that knockdown of PSAP blocked DR6-induced Bax translocation and cytochrome c release from the mitochondria. Moreover, it was found that both PSAP and DR6 form complexes with Bax, but at different subcellular locations. The DR6-Bax complex was detected in the cytosolic fraction while the PSAP-Bax complex was detected in the mitochondrial fraction. The observation that knockdown of DR6 significantly reduced the amount of PSAP-Bax complex detected in mitochondria suggests a possibility that DR6-bound Bax is transferred to PSAP upon interaction with PSAP at the mitochondria, leading to cytochrome c release and eventually apoptosis.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription–quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options The work in GAC’s laboratory was partly supported by NIH/National Center for Advancing Translational Sciences grant UH3TR00943-01, the NIH/National Cancer Institute (NCI) grant 1 R01 CA182905-01, U54 grant UPR/MDACC Partnership for Excellence in Cancer Research 2016 Pilot Project, Team Department of Defense grant CA160445P1, a Ladies Leukemia League grant, a Chronic Lymphocytic Leukemia Moonshot Flagship project, a Sister Institution Network Fund 2017 grant, and the Estate of C. G. Johnson, Jr. EFM was supported in part by award number P50 CA140388 from the NCI and by the NIH Clinical Research Loan Repayment Program. AMS was supported by Fundação para a Ciência e a Tecnologia through fellowship SFRH/BD/85968/2012. ZJZ’s work was supported by grants from the MPN Foundation and the Oklahoma Center for the Advancement of Science and Technology. DG, MPD, and IBN were supported in part by a Programul Opera¿ional Competitivitate grant nr35/01.09.2016, ID 37_796, titled “Clinical and economical impact of personalized targeted anti-mi-croRNA therapies in reconverting lung cancer chemoresistance” (CANTEMIR). NSDA was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, BEPE 2016/09349-4.

2019-nCoV is a newly identified coronavirus with high similarity to SARS-CoV. We performed a structural analysis of the receptor binding domain (RBD) of spike glycoprotein responsible for entry of coronaviruses into host cells. The RBDs from the two viruses share 72% identity in amino acid sequences, and molecular simulation reveals highly similar ternary structures. However, 2019-nCoV has a distinct loop with flexible glycyl residues replacing rigid prolyl residues in SARS-CoV. Molecular modeling revealed that 2019-nCoV RBD has a stronger interaction with angiotensin converting enzyme 2 (ACE2). A unique phenylalanine F486 in the flexible loop likely plays a major role because its penetration into a deep hydrophobic pocket in ACE2. ACE2 is widely expressed with conserved primary structures throughout the animal kingdom from fish, amphibians, reptiles, birds, to mammals. Structural analysis suggests that ACE2 from these animals can potentially bind RBD of 2019-nCoV, making them all possible natural hosts for the virus. 2019-nCoV is thought to be transmitted through respiratory droplets. However, since ACE2 is predominantly expressed in intestines, testis, and kidney, fecal-oral and other routes of transmission are also possible. Finally, antibodies and small molecular inhibitors that can block the interaction of ACE2 with RBD should be developed to combat the virus.

Juvenile myelomonocytic leukemia (JMML) is a highly fatal malignant disease in early childhood. It is still unknown of the specific pathogenesis, and there is shortage of effective targeted therapeutic approaches. Gain of function SHP2 mutation encoded by PTPN11 gene is found in approximately 35% of JMML patients, which maybe contributed to its pathogenesis. JMML patients with SHP2 mutation have lower survival rate and higher recurrence rate. All of the above make development of new therapies imperative. Currently, there is no stable cell line that can accurately reflect the characteristics of JMML abnormal cells for research on JMML. In this study, we established two leukemia cell lines that depend on mutated SHP2 for survival, and discovered promising drugs that targeted mutated-SHP2-dependent oncogenic signaling pathway through drug screening method. HCD-57 cells are murine erythroleukemia cells that solely depend on exogenic erythropoietin (EPO) for survival. We constructed SHP2-D61Y and SHP2-E76K transformed HCD-57 cell lines through retroviral vectors, the survival of which dependent on mutated SHP2 mediated signaling pathway. Based on these cells, we established a drug screening platform and screened small molecule compound library containing 2862 FDA-approved drugs and 1707 kinase inhibitors. We performed cell viability, flow cytometry, Wright-Giemsa staining, and western blot to evaluate cells after drug treatment. To further assess therapeutic potential, we established in-vivo transplantation model that SHP2-D61Y transformed HCD-57 cells were implanted into immunodeficient NCG mice, and verified the effectiveness of the in-vitro screened drugs. We found that the survival and proliferation of HCD-57 cells transduced by SHP2-D61Y and SHP2-E76K no longer required EPO, but completely relied on the abnormal activation of signaling pathway mediated by mutated SHP2. Western blot results showed that the phosphorylation status of ERK1/2 and AKT of HCD-57 cells expressing SHP2 mutation were abnormally increased, consistent with SHP2-mutated JMML. Thus, we have obtained the leukemia cell lines that can represent the characteristics of activated signaling pathway in JMML with SHP2 mutation. Through drug screening, we observed that drug sunitinib (Sutent ®) selectively inhibits SHP2-mutated HCD-57 cell lines. CCK-8-based cell viability assay demonstrated a dose-dependent inhibition of SHP2-D61Y and SHP2-E76K transformed HCD-57 cell and no effects on the parental HCD-57 cells. Live cell counting with trypan blue revealed that the proliferation of SHP2-mutated HCD-57 cells was totally halted after one day upon treatment with 250 nM sunitinib, whereas the HCD-57 cells were unaffected. Wright-Giemsa staining demonstrated that SHP2-mutated HCD-57 cells showed no normal morphology change and no mitotic activity under sunitinib treatment, otherwise parental HCD-57 cells showed normal mitotic activity. Sunitinib induced apoptosis and cell cycle arrest at G1 phase in SHP2-mutated HCD-57 cells by flow cytometry, but had little effect on the parental HCD-57 cells. Sunitinib effectively downregulates the phosphorylation of ERK and AKT in SHP2-mutated cells, revealing the mechanism of sunitinib targeting SHP2-mutated cells. In addition, after transplantation of SHP2-D61Y transformed HCD-57 cells for 3 weeks, the spleen of NCG mice increased from an average of 45 mg to more than 300 mg; flow cytometry analysis showed that the implanted cells accounted for over 75% of the total nucleated cells in the bone marrow and spleen. Compared with the vehicle control, the number of monocytes in these mice was reduced to the normal range by treatment with sunitinib, and the spleen weights were reduced by about 50%. Histochemical staining showed disappearance of the myeloid infiltration in the spleen, liver and bone marrow. The above results all indicate that sunitinib has strong in-vivo anti-leukemia activity. Furthermore, western blot analysis showed that the administration of sunitinib significantly inhibited the phosphorylation expression level of AKT and ERK, indicating the effectivity of sunitinib in vivo. In conclusion, our data demonstrated that HCD-57 cell line is an effective tool for studying oncogenic signaling pathway and screening drugs that targeted JMML with SHP2 mutation. Sunitinib can be an effective drug for the targeted treatment of JMML in the future. Disclosures No relevant conflicts of interest to declare.

Protein tyrosine phosphatase SHP2 encoded by PTPN11 is a key regulator in growth factor and cytokine signaling. Overwhelming evidence suggests its vital role in hematopoietic stem cell function and hematopoiesis. As a bona fide proto-oncogene product, gain-of-function mutations of SHP2 cause hematological malignancies, most notably juvenile myelomonocytic leukemia (JMML) which bear somatic SHP2 mutations in 35% of cases. Numerous studies have utilized murine models to investigate the role of mutant SHP2 in hematopoiesis and leukemogenesis and successfully produced resembling myeloproliferative neoplasm (MPN) and even full-blown leukemia in recipient animals. However, mutant SHP2-transformed cell lines have not been generated. In the present study, we established oncogenic mutant SHP2-transformed cell lines from erythropoietin (EPO)-dependent HCD-57 erythroid leukemia cells. First, we generated recombinant retroviruses expressing SHP2-D61Y and SHP2-E76K, the two most common SHP2 mutants found in individuals with JMML, by using the pMSCV-IRES-GFP vector. We then infected HCD-57 cells with the recombinant retroviruses. Unlike the parent HCD-57 cells, the infected cells were able to grow in the absence of EPO as demonstrated by viable GFP-positive cells. We further performed semi-solid methylcellulose colony cultures and isolated single clones of EPO-independent HCD57 cells. The isolated clonal cells overexpressed mutant SHP2 and proliferate rapidly in the absence of EPO. In contrast, HCD57 cells infected with retroviruses expressing wild type SHP2 failed to survive in the absence of EPO, indicating only gain-of-function mutant forms of SHP2 have the cell-transformation capability. We also carried out parallel experiments with the pro-B Ba/F3 cell line that require interleukin 3 (IL3) for survival. Interestingly, over-expression of SHP2-D61Y and SHP2-E76K was not sufficient to give rise to IL3-indepdent Ba/F3 cells, suggesting that HCD57 cells have some unique properties making them susceptible for transformation by oncogenic SHP2 mutants. We further performed in vitro and in vivo characterization of transformed HCD57 cells. Cell signaling analyses revealed that both HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells exhibited aberrantly elevated levels of pERK and pAKT in the absence of cytokine stimulation, which was consistent with the notion that gain-of-function SHP2 mutants perturb growth control through deregulation of the Ras signaling pathway. Upon intravenous injection into immunodeficient mice, the SHP2 mutant-transformed HCD57 cells caused acute leukemia with markedly increased spleen. Finally, we screened a small molecule inhibitor library to identify compounds that may specifically target the SHP2 mutants. We found several tyrosine kinase inhibitors including dasatinib and trametinib potently inhibited HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells but not the parent HCD57 cells. At sub-micromolar concentrations, dasatinib and trametinib abolished elevated ERK and Akt activation caused by the SHP2 mutants. This study not only proves that gain-of function mutations of SHP2 are capable of fully transforming cells but also provides a unique cell system to study pathogenesis of SHP2 mutants and to identify specific inhibitors for drug development. Disclosures No relevant conflicts of interest to declare.

Study Objective To compare the distribution of initial procedures performed as treatment of uterine fibroids between the different classes of patient body mass index (BMI). Design Retrospective cohort study. Setting Academic medical center. Patients or Participants We included patients (N=1982) with known fibroids (based on ICD codes and ultrasound findings) who underwent a fibroid procedural intervention between 2008 and 2015. Interventions Uterine fibroid embolization (UFE), myomectomy (excluding hysteroscopic procedures), or hysterectomy Measurements and Main Results The main exposure was patient BMI class: normal weight (n=498), overweight (n=518), and class I (n=413), II (n=260), and III (n=293) obesity. The main outcome was the first fibroid procedural intervention (UFE, myomectomy, or hysterectomy) performed during the study time period. Amongst women of normal weight who underwent a fibroid procedural intervention, 67.5% of the time this procedure was hysterectomy. Comparatively, overweight women and women with class I, II, and III obesity underwent hysterectomy as their initial procedure 71.4%, 77.7%, 78.1%, and 78.8% of the time (p Multivariable logistic regression adjusted for patient age, race, and the presence of the obesity-related comorbidities of hypertension, diabetes, and obstructive sleep apnea. When compared to women of normal weight, women with class I (aOR 1.8, 95% CI 1.3-2.5, p Conclusion Obese women were significantly more likely to undergo hysterectomy as their initial fibroid procedural intervention as opposed to uterine-preserving procedures, even when adjusted for key factors that may influence a patient's and surgeon's decision regarding operative management. Future studies elaborating on indication for surgery and fibroid burden may shed light on this phenomenon.

Study Objective To assess the relationship between patient body mass index (BMI) and operating room (OR) scheduling inaccuracies at the time of hysterectomy. Design Retrospective cohort study. Setting Academic medical center. Patients or Participants Scheduled hysterectomies (n=951) performed from 11/2017 through 02/2020 for any gynecologic indication. Interventions Hysterectomy by any route. Measurements and Main Results The primary outcome was operating room time scheduling inaccuracy in minutes, defined as the difference between the scheduled and the actual operating room time. The mean scheduling inaccuracy for women with normal weight, overweight, and class I, II, and III obesity was 22.2, 28.4, 33.5, 38.3, and 48.2 minutes, respectively (p Multivariable linear regression analysis adjusted for patient age, the presence of major cardiopulmonary comorbidities, ASA category, anesthetic type, abdominal surgical history, number of procedures performed, procedure route, and uterine weight. Normal and overweight women were similarly under-scheduled. Compared with normal-weight women, those with class 1 and 3 obesity were, on average, 12.7 (95% CI 2.1-23.3, p=0.019) and 18.9 (95% CI 4.4-33.3, p=0.010), respectively, more minutes under-scheduled. (The average scheduling inaccuracy of women with class 2 obesity did not differ significantly from women with normal weight.) Obese women whose surgeries were under-scheduled, compared to those who were not, spent a significantly greater proportion of their operating room time in operative time (82.6% vs. 74.6%, p Conclusion When adjusted for key factors that may affect operating room time, obese women were significantly more under-scheduled than women of normal weight. Obese women with under-scheduled cases spent proportionally more OR time in operative as opposed to non-operative time. Future studies can assess whether awareness of this tendency helps surgeons more accurately predict operating room time required for hysterectomy for obese women.

Recognition of major histocompatibility complex (MHC) class I antigens on tumor cells by cytotoxic T cells is involved in T cell-mediated tumor immune surveillance and immune checkpoint therapy. The interferon-γ (IFNγ)-IRF1 signaling pathway regulates MHC class I antigen presentation. To examine genetic defects of the IFNγ-IRF1 pathway in non-small cell lung cancer (NSCLC), we analyzed The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LuAd) and lung squamous cell carcinoma (LuSc) data. Loss-of-function (LOF) genetic alterations of the IFNγ-IRF1 pathway genes (IFNGR1, IFNGR2, JAK1, JAK2, STAT1, IRF1) were found in 64 (6.3%) of 1,016 patients. These genetic defects occur prevalently in JAK2 (33 cases) and often through deletions (29 cases) of chromosome 9p24.1. JAK2 deletions were frequently, but not always, associated with deletions of PD-L1 gene (CD274), PD-L2 gene (PDCD1LG2), PTPRD, and CDKN2A/CDKN2B at the chromosome 9p24.1-9p21.3 region. IRF1 expression was correlated with immune cytolytic activity markers GZMA and PRF1 in NSCLC. IFNγ induced IRF1 expression and cell surface HLA-A/HLA-B/HLA-C (HLA-ABC) in A549, H661, H292, and H2172 cells that contained the wildtype JAK2, but not in H1573 and H1623 cells that were JAK2 defective. Deletion of JAK2 or inhibition of the JAK2 kinase activity resulted in loss of IFNγ-induced IRF1 and cell surface HLA-ABC in JAK2 wildtype NSCLC cells, whereas expression of exogenous JAK2 in H1573 cells restored the IFNγ responses. These findings show that JAK2 deficiency is the major mechanism of genetic defects of the IFNγ-IRF1 pathway in NSCLC and reveal a previously unrecognized significance of chromosome 9p deletion in NSCLC.

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of B lymphocytes blocked at an early stage of differentiation, which accounts for approximately 30% of childhood cancers. Almost all B-ALL blasts aberrantly express FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase involved in the development of dendritic cells and B lymphocytes. In B-ALL, the high expression of FLT3 in blasts significantly increases the odds of relapse/death in patients, indicating a pathological role of FLT3 in B-ALL. Note that gain-of-function mutations of FLT3 are relatively rare ( Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a hematological malignancy featured by impaired differentiation and uncontrolled proliferation of myeloid blasts. FLT3 internal tandem duplication (ITD) presents in 30-40% patients with AML, which serves as an independent poor prognostic marker and an attractive therapeutic target. Up to date, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved by FDA in the treatment of AML. However, FLT3 TKIs as single agents have limited effects to eliminate AML cells due to multiple drug-resistant mechanisms, including secondary FLT3 mutations, alternatively activated cell survival pathways, upregulation of FLT3 ligand, and downregulation of tumor suppressor genes. In the present study, we found that FLT3 TKIs decreased tumor suppressor p53 protein level by downregulation of YOD1 through miR-181 in FLT3-ITD mutant AML cells. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. By using transcriptome analysis with RNAseq, we identified multiple differentially expressed miRNAs in HCD-57 transformed by FLT3-ITD, compared with parental HCD-57 cells. miR-181a-5p and miR-181b-5p were among these highly differentially expressed miRNAs. These two miRNAs were predicted to bind in 3'UTR of deubiquitinase YOD1 by using TargetScanHuman 7.2, an online tool to predict biological targets of miRNAs. Previous studies have shown that the ubiquitination and protein level of p53 is dysregulated due to overexpressed E3 ligase MDM2/4 in AML cells, but few studies focused on deubiquitinase of p53. We found that deubiquitinase YOD1 interacted with p53 by immunoprecipitation. Overexpression of YOD1 prevented degradation of p53 led by cycloheximide, a protein synthesis inhibitor. We further found that overexpression of YOD1 resulted in decreased ubiquitination of p53, indicating that YOD1 stabilized p53 protein through deubiquitination. Subsequently, we detected the expression of miR-181, YOD1 and p53 in a FLT3-ITD positive AML cell line MV-4-11 treated by FLT3 TKIs sorafenib, sunitinib and quizartinib. Real-time quantitative PCR showed that the treatment of FLT3 TKIs upregulated the expression of miR-181a-5p/miR-181b-5p, and downregulated mRNA level of their predicted target YOD1. The mRNA level of p53 remained unchanged but its protein level decreased with enhanced ubiquitination in MV-4-11 cells treated by FLT3 TKIs in the ubiquitination assay. These data suggested that FLT3 TKIs could reduce the stability of p53 by regulating miRNA-targeted YOD1. In addition, we collected peripheral blood mononuclear cells (PBMCs) from patients with AML and age-matched healthy donors. We found increased expression of miR-181a-5p and miR-181b-5p and decreased expression of YOD1 in PBMCs from AML patients compared with healthy donors, suggesting a pathological role of miR-181 to regulate YOD1/53 pathway in AML. In conclusion, our data showed that FLT3 TKIs induced instability of p53 by miR-181 mediated downregulation of YOD1. YOD1 as a novel deubiquitinase of p53 could play important roles in drug-resistance and progression of AML. Disclosures No relevant conflicts of interest to declare.

FLT3 internal tandem duplication (FLT3-ITD) is an activating mutation found in 20-30% of patients with acute myeloid leukemia (AML), which makes FLT3 an attractive target for the treatment of AML. Although FLT3-mutant patients respond to current FLT3 inhibitors, relapse usually happens because of the acquisition of resistant secondary mutations at the FLT3 catalytic domain, which is mainly on D835. In the search for compounds with broad FLT3 inhibition activities, we screened a kinase inhibitor library by using our unique FLT3 substrate and identified JAK3 inhibitor VI (designated JI6 hereafter) as a novel FLT3 inhibitor, which selectively targets FLT3 D835 mutants as well as FLT3-ITD. JI6 effectively inhibited FLT3-ITD-containing MV4-11 cells and HCD-57 cells transformed with FLT3-ITD and D835 mutants. Furthermore, administration of JI6 effectively targeted FLT3 signaling in vivo and suppressed the myeloproliferative phenotypes in FLT3-ITD knock-in mice, and significantly prolonged the survival of immunodeficient mice implanted with the transformed HCD-57 cells. Therefore, JI6 is a promising candidate for the development of next-generation anti-AML drugs.

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

To examine the impact of not blinding outcome assessors on estimates of intervention effects in animal experiments modeling human clinical conditions.We searched PubMed, Biosis, Google Scholar, and HighWire Press and included animal model experiments with both blinded and nonblinded outcome assessors. For each experiment, we calculated the ratio of odds ratios (ROR), that is, the odds ratio (OR) from nonblinded assessments relative to the corresponding OR from blinded assessments. We standardized the ORs according to the experimental hypothesis, such that an ROR1 indicates that nonblinded assessor exaggerated intervention effect, that is, exaggerated benefit in experiments investigating possible benefit or exaggerated harm in experiments investigating possible harm. We pooled RORs with inverse variance random-effects meta-analysis.We included 10 (2,450 animals) experiments in the main meta-analysis. Outcomes were subjective in most experiments. The pooled ROR was 0.41 (95% confidence interval [CI], 0.20, 0.82; I(2) = 75%; P 0.001), indicating an average exaggeration of the nonblinded ORs by 59%. The heterogeneity was quantitative and caused by three pesticides experiments with very large observer bias, pooled ROR was 0.20 (95% CI, 0.07, 0.59) in contrast to the pooled ROR in the other seven experiments, 0.82 (95% CI, 0.57, 1.17).Lack of blinding of outcome assessors in animal model experiments with subjective outcomes implies a considerable risk of observer bias.

Pruritus occurs frequently in patients with polycythemia vera (PV), and the pathophysiology of PV-associated pruritus is unclear. We have previously demonstrated that transgenic mice expressing JAK2V617F displayed clear PV-like phenotypes. In the current study, we found frequent occurrence of pruritus with aged JAK2V617F transgenic mice and further investigated the underlying mechanisms by studying mast cells, key players in allergic reactions and anaphylaxis. Massive accumulations of mast cells were observed in the skin of pruritic JAK2V617F transgenic mice. In vitro culture yielded much higher mast cell counts from the bone marrow, spleen, peripheral blood, and peritoneal cavity of JAK2V617F transgenic mice than from controls. Cultured mast cells from JAK2V617F transgenic mice exhibited enhanced proliferative signals, relative resistance to cell death upon growth factor deprivation, and a growth advantage over control cells under suboptimal growth conditions. However, these mast cells displayed normal morphology and contained normal levels of mast cell proteases before and after degranulation. Finally, the JAK2 inhibitor G6 effectively reduced mast cell numbers and alleviated pruritus in JAK2V617F transgenic mice. Collectively, these data demonstrate that mast cells are involved in PV-associated pruritogenesis and that JAK2 inhibitors are potential antipruritus drugs.

Platelets are crucial for normal hemostasis; however, their hyperactivation also contributes to many potentially lethal pathologies including myocardial infarction, stroke, and cancer. We hypothesized that modified platelets lacking their aggregation and activation capacity could act as reversible inhibitors of platelet activation cascades. Here, we describe the development of detergent-extracted human modified platelets (platelet decoys) that retained platelet binding functions but were incapable of functional activation and aggregation. Platelet decoys inhibited aggregation and adhesion of platelets on thrombogenic surfaces in vitro, which could be immediately reversed by the addition of normal platelets; in vivo in a rabbit model, pretreatment with platelet decoys inhibited arterial injury-induced thromboembolism. Decoys also interfered with platelet-mediated human breast cancer cell aggregation, and their presence decreased cancer cell arrest and extravasation in a microfluidic human microvasculature on a chip. In a mouse model of metastasis, simultaneous injection of the platelet decoys with tumor cells inhibited metastatic tumor growth. Thus, our results suggest that platelet decoys might represent an effective strategy for obtaining antithrombotic and antimetastatic effects.

A role for macrophages in erythropoiesis was suggested several decades ago when erythroblastic islands in the bone marrow, composed of a central macrophage surrounded by developing erythroblasts, were described. However, the in vivo role of macrophages in erythropoiesis under homeostatic conditions or in disease remains unclear. We found that specific depletion of CD169(+) macrophages markedly reduced the number of erythroblasts in the bone marrow but did not result in overt anemia under homeostatic conditions, probably because of concomitant alterations in red blood cell clearance. However, CD169(+) macrophage depletion significantly impaired erythropoietic recovery from hemolytic anemia, acute blood loss and myeloablation. Furthermore, macrophage depletion normalized the erythroid compartment in a JAK2(V617F)-driven mouse model of polycythemia vera, suggesting that erythropoiesis in polycythemia vera remains under the control of macrophages in the bone marrow and splenic microenvironments. These results indicate that CD169(+) macrophages promote late erythroid maturation and that modulation of the macrophage compartment may be a new strategy to treat erythropoietic disorders.

Background microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. Methods Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Results Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. Conclusions Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0753-x) contains supplementary material, which is available to authorized users.

Through exome resequencing, we identified two unique mutations in recombination signal binding protein for immunoglobulin kappa J (RBPJ) in two independent families affected by Adams-Oliver syndrome (AOS), a rare multiple-malformation disorder consisting primarily of aplasia cutis congenita of the vertex scalp and transverse terminal limb defects. These identified mutations link RBPJ, the primary transcriptional regulator for the Notch pathway, with AOS, a human genetic disorder. Functional assays confirmed impaired DNA binding of mutated RBPJ, placing it among other notch-pathway proteins altered in human genetic syndromes.

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several hematological disorders, including myeloproliferative neoplasms. Effective Jak2 inhibitors can have significant therapeutic potential. Here, using structure-based virtual screening, we identified a benzothiophene-derived Jak2 inhibitor named A46 . We hypothesized that this compound would inhibit Jak2-V617F–mediated pathologic cell growth. To test this, A46 was analyzed for its ability to inhibit recombinant Jak2 protein catalysis; suppress Jak2-mediated pathogenic cell growth in vitro; inhibit the aberrant ex vivo growth of Jak2-V617F–expressing primary human bone marrow cells; and inhibit Jak2-mediated pathogenesis in vivo. To this end, we found that A46 selectively inhibited Jak2-V617F protein when compared to wild-type Jak2 protein. The drug also selectively inhibited the proliferation of Jak2-V617F–expressing cells in both a time- and dose-dependent manner, and this correlated with decreased Jak2 and signal transducers and activators of transcription 5 phosphorylation within treated cells. The Jak2-V617F cell growth inhibition correlated with an induction of cell cycle arrest and promotion of apoptosis. A46 also inhibited the pathologic growth of primary Jak2-V617F–expressing bone marrow cells ex vivo. Lastly, using a mouse model of Jak2-V617F–mediated myeloproliferative neoplasia. A46 significantly reduced the splenomegaly and megakaryocytic hyperplasia in the spleens of treated mice and the levels of interleukin-6 in the plasma. Collectively, our data demonstrate that the benzothiophene-based compound, A46, suppresses Jak2-mediated pathogenesis, thereby making it a potential candidate drug against Jak2-mediated disorders.

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although molecular mechanisms driving disease progression remain elusive. While most patients who acquire a JAK2V617F mutation in CD34+ cells present with chronic, indolent Polycythemia Vera (PV), ~25% will progress to MF or AML. High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive leukemia where they portend adverse outcomes. In murine models, Hmga1/2 overexpression drives clonal expansion and uncontrolled proliferation. HMGA1/2 genes are also overexpressed in MPN with disease progression. We therefore sought to: 1) test the hypothesis that HMGA proteins are required for leukemic transformation and rational therapeutic targets in MPN progression, and, 2) identify mechanisms mediated by HMGA1/2 during disease progression. Methods: We measured HMGA1/2 in JAK2V617F mutant human AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV; Blood 2015;126:484). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cell lines (DAMI, SET-2) and assessed proliferation, colony formation, and leukemic engraftment in immunodeficient mice. To further assess Hmga1 function in vivo, we crossed mice with heterozygous Hmga1 deficiency onto murine models of PV and PV-AML. Finally, to dissect molecular mechanisms underlying HMGA1, we compared RNA-Seq from MPN-AML cell lines (DAMI, SET-2) after silencing HMGA1/2 to that of controls and applied Ingenuity Pathway Analysis. Results: HMGA1/2 mRNA are up-regulated in all JAK2V617F-positive contexts, including primary human PV CD34+ cells and total bone marrow from JAK2V617F mouse models for PV compared to controls. Further, there is a marked up-regulation in both HMGA1/2 in CD34+ cells from PV patients after transformation to MF or AML and in leukemic blasts from our PV-AML mouse model compared to PV mice. Overexpression of HMGA1/2 also correlates with clonal dominance of human JAK2V617F-homozygous stem cells and additional mutations of epigenetic regulators (EZH2, SETBP1). Silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemic engraftment in mice. Further, heterozygous Hmga1 deficiency decreases splenic enlargement in PV mouse models with advancing age. Moreover, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality. PV-AML mice survived a median of 5 weeks whereas PV-AML mice with heterozygous Hmga1 deficiency survive a median of 12 weeks (P< 0.002). The leukemic burden was also decreased in mice with Hmga1 deficiency. Preliminary RNA-Seq analyses from DAMI and SET-2 cells show that HMGA1 drives pathways involved in Th1/Th2 activation, chemotaxis, cell-cell signaling, myeloid cell accumulation and other immune cell trafficking, inflammation, and injury, suggesting that HMGA1 co-opts immune and inflammatory networks to drive tumor progression. Surprisingly, atherosclerosis pathways are also induced by HMGA1. Conclusions: HMGA1/2 genes are overexpressed in MPN with highest levels in more advanced disease (MF, AML) both in primary human tumors and murine models. Strikingly, silencing HMGA1 or HMGA2 halts proliferation and clonogenicity in vitro and prevents leukemic engraftment in vivo. Further, heterozygous Hmga1 deficiency prolongs survival in a murine model of fulminant MPN AML and decreases tumor burdens. Finally, preliminary RNA-Seq analyses suggest that HMGA1 amplifies transcriptional networks involved in immune cell trafficking and inflammation to drive tumor progression. Unexpectedly, HMGA1 also regulates pathways involved in atherosclerosis, implicating HMGA1 as a novel link between clonal hematopoiesis and cardiovascular disease. Our findings further highlight HMGA1/2 as a key molecular switch for leukemic transformation in MPN and opens the door to novel therapeutic approaches to prevent disease progression. Disclosures No relevant conflicts of interest to declare.

The Thrombopoietin (THPO)/ thrombopoietin receptor (MPL) signaling axis is not only critical for the generation of platelets and megakaryocytes, but also for the maintenance of hematopoietic stem cells (HSC) and the bone marrow niche. MPL is expressed on primitive HSC, HSC progenitors, megakaryocytes, platelets, osteoblasts and osteoclasts, clonal hematopoietic stem cells and many leukemias. THPO production is constitutive but is also increased by inflammatory cytokines. Sustained exposure to high levels of THPO not only enhances platelet production, but also has a profound effect on HSC and the bone marrow microenvironment. Excess THPO/MPL signaling, whether driven by inflammatory cytokines, or due to mutations in THPO, JAK2, MPL or CALR, is associated with HSC expansion, megakaryocyte hypertrophy and increased platelet count, excess release of megakaryocyte and platelet-based cytokines such as TGF-beta and PDGF-alpha, and the development of stromal myofibroblasts that drive tissue fibrosis and anemia. We developed a robust and clinically validated RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent N-acetylgalactosamine (GalNAc) conjugates, enabling specific silencing of hepatocyte-expressed genes following subcutaneous injection. Since liver is the major source of THPO expression, we utilized GalNAc-siRNA technology to develop siRNAs targeting THPO for evaluation in wild type mice and murine models of myeloproliferative neoplasms (MPN). Active siRNAs were identified by in vitro screening in primary mouse hepatocytes and the 12 best siRNAs were evaluated in vivo in normal mice to select the most potent siRNA. THPO liver mRNA levels were reduced by up to 80% after a single subcutaneous THPO siRNA dose, with no effect on THPO mRNA expression in other organs (kidney, and bone marrow, both of which had marginal THPO expression compared to liver). Circulating TPO levels were reduced by 80% by day 7 and were suppressed for up to 28 days post a single dose treatment. Platelet counts were reduced to 60% of baseline by day 14, and a further reduction to more than 70% of baseline was observed with every other week dosing. No changes in red blood cell or white blood cell subsets were observed. Platelet reduction was accompanied by a reduction in megakaryocyte mass, as evidenced by a 50% decrease in the number of bone marrow megakaryocytes in THPO siRNA treated mice compared to controls. Mice treated every other week with TPO siRNA for three months demonstrated sustained circulating THPO protein and platelet count reductions, and a significant reduction in bone marrow HSC, Lin-Sca1+Kit- (LSK) and multipotent progenitor (MPP) frequency. Evaluation of impact THPO silencing on MPN phenotypes in transgenic JAK2V617F mice is ongoing. THPO silencing is a potential novel targeted therapeutic approach that may be beneficial in benign and malignant conditions in which deregulated THPO/MPL signal transduction drives disease pathology. Disclosures No relevant conflicts of interest to declare.

The JAK2V617F mutation was found in most patients with myeloproliferative disorders (MPDs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2V617F, showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2V617F expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2V617F can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2V617F and to develop treatment for MPDs.

JAK2(V617F), a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva) is a potent inhibitor of JAK2(V617F) activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2(V617F)-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2(V617F)-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2(V617F)-positive PV and other myeloproliferative disorders.

MicroRNAs (miRNAs) are important epigenetic regulators of gene expression. Although several miRNAs have been implicated in osteosarcoma, their role in regulation of osteosarcoma cancer stem cells (CSCs) remains unknown. Here we demonstrated that miR-26a is downregulated in osteosarcoma CSCs when derived by either sarcosphere generation, chemodrug or aldehyde dehydrogenase (ALDH) activity selection. Lentiviral overexpression of miR-26a in ZOS and 143B osteosarcoma cells decreases the expression of stem cell markers and suppresses sarcosphere formation, as well as ALDH activity. Moreover, miR-26a overexpression inhibits the tumor cell growth both in vitro and in vivo. We further demonstrate that miR-26a directly target Jagged1, one of the Notch ligand, and that its tumor suppressive effects are mediated through inhibition of Jagged1/Notch signaling. Importantly, reduced miR-26a expression, as determined by in situ hybridization in patient tumors (n=92), is associated with lung metastasis and poor overall survival of osteosarcoma patients. Together, these data suggest the essential role of miR-26a/Jagged1/Notch pathway in regulating the stem cell-like traits of osteosarcoma cells and provide a potential target for osteosarcoma therapy.

The Ten-eleven translocation (TET) family proteins are evolutionarily conserved dioxygenases responsible for the conversion of 5-methylcytosine to 5-hydroxymethylcytosine, thereby promoting DNA demethylation. There are three members in this family, namely TET1, TET2, and TET3. They contain structural features conferring unique substrate specificity and cofactor requirements and are subject to transcriptional control by transcription factors and microRNAs, and to posttranslational regulation by protein modifications and interacting proteins. TET2 is highly expressed in hematopoietic cells and plays a key role in hematopoiesis by controlling hematopoietic stem cell self-renewal, lineage commitment, and terminal differentiation. Analysis of TET2 knockout mice demonstrated a tumor suppressor function of TET2 whose loss and haplo-insufficiency initiate myeloid and lymphoid malignancies. TET2 inactivation, through loss-of-function mutations and isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations, is a common event in hematopoietic malignancies. Understanding the structure, regulation, and function of TET2 should provide a better strategy to treat hematological malignancies.

MEG2, a protein tyrosine phosphatase with a unique NH2-terminal lipid-binding domain, binds to and is modulated by the polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3. Recent data implicate MEG2 in vesicle fusion events in leukocytes. Through the genesis of Meg2-deficient mice, we demonstrate that Meg2−/−embryos manifest hemorrhages, neural tube defects including exencephaly and meningomyeloceles, cerebral infarctions, abnormal bone development, and >90% late embryonic lethality. T lymphocytes and platelets isolated from recombination activating gene 2−/− mice transplanted with Meg2−/− embryonic liver–derived hematopoietic progenitor cells showed profound defects in activation that, in T lymphocytes, was attributable to impaired interleukin 2 secretion. Ultrastructural analysis of these lymphocytes revealed near complete absence of mature secretory vesicles. Taken together, these observations suggest that MEG2-mediated modulation of secretory vesicle genesis and function plays an essential role in neural tube, vascular, and bone development as well as activation of mature platelets and lymphocytes.

Protein tyrosine kinases (PTKs) and phosphatases (PTPs) play a crucial role in normal cell development, and dysfunction of these enzymes has been implicated in human cancers. Polycythemia vera (PV) is a clonal hematologic disease characterized by hypersensitivity of hematopoietic progenitor cells to growth factors and cytokines. Recently, a unique and clonal mutation in the JAK homology 2 (JH2) domain of JAK2 that results in a valine to phenylalanine substitution at position 617 (V617F) was found in the majority of PV patients. This mutation leads to constitutive JAK2 activation and abnormal signaling and induces erythrocytosis in an animal model. The mutation is also found in a significant percentage of patients with idiopathic myelofibrosis (50%) and essential thrombocythemia (30%). Thus, it seems probable that this mutation associates with other molecular genetic events to cause different myeloproliferative disorders (MPDs). One of these secondary events is the transition to homozygosity of the mutated gene in 30% of the PV patients. Other events may include defects in PTPs, but these remain to be characterized. Recent studies represent a great step forward in the molecular pathogenesis in PV and the development of targeted new drugs to treat the disease.

Mutations of the polycystic kidney and hepatic disease 1 ( PKHD1 ) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct–lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

Polycythemia vera (PV) is a human clonal hematologic disorder. Previously we demonstrated that erythroid colony-forming cells (ECFCs) from PV patients contained a hyperactive membrane-associated tyrosine phosphatase. We now show that this phosphatase corresponded to protein tyrosine phosphatase (PTP)-MEG2, an intracellular enzyme with a putative lipid-binding domain. The increased activity of PTP-MEG2 in PV cells is due to its elevated distribution in the membrane fraction. With the development of ECFCs to mature red cells, the protein level of PTP-MEG2 decreased gradually, but membrane-associated PTP-MEG2 was sustained for a longer period of time in PV cells, which correlated with an enhanced colony-forming capability of the cells. Importantly, expression of dominant-negative mutant forms of PTP-MEG2 suppressed in vitro growth and expansion of both normal and PV ECFCs. The data indicate that PTP-MEG2 has an important role in the development of erythroid cells. (Blood. 2003;102:4354-4360)

Protein tyrosine phosphatase (PTP)-MEG2 is an intracellular tyrosine phosphatase that contains a Sec14 homology domain. We have purified the full-length and truncated forms of the enzyme from recombinant adenovirus-infected human 293 cells. By using lipid-membrane overlay and liposome binding assays, we demonstrated that PTP-MEG2 specifically binds phosphatidylserine among over 20 lipid compounds tested. The binding is mediated by its N-terminal Sec14 domain. In intact cells, the Sec14 domain is responsible for localization of PTP-MEG2 to the perinuclear region, and uploading of PS into the cell membrane causes translocation of PTP-MEG2 to the plasma membrane. Phosphatidylserine is a relatively abundant cell membrane phospholipid non-symmetrically distributed in the outer layer and inner layer of cell membranes. It has recently been defined as an important ligand for clearance of apoptotic cells. By specifically binding phosphatidylserine, PTP-MEG2 may play an important role in regulating signaling processes associated with phagocytosis of apoptotic cells.

Myelofibrosis (MF) is a common feature of the Philadelphia-Chromosome negative (Ph-) myeloproliferative neoplasms (MPN), a group that includes Primary Myelofibrosis (PMF), Essential Thrombocytosis (ET) and Polycythemia Vera (PV). Current treatment with Jak2 inhibitors reduces splenomegaly, but does not eliminate malignant clones nor improve bone marrow (BM) or spleen fibrosis, underlining the need for alternative therapies. One promising target is TGF-β1, a cytokine crucial for the development of myelofibrosis that is elevated in PMF patient plasma, progenitors and megakaryocytes (MK), and is known to promote fibrosis through stimulation of fibroblasts and their progenitors, mesenchymal stroma cells (MSC). Dysregulation of TGF-β1 signaling has been described in a number of mouse models of myelofibrosis exhibiting aberrant megakaryopoiesis, one of the hallmarks of MPNs. Preliminary data from 93 MPN patients showed overexpression of TGF-ß in PMF samples (p=0.001), with increased activation of the canonical target SMAD2 (p=0.005). We tested the efficacy of the orally available TGF-β1 signaling inhibitor Galunisertib (LY2157299) in counteracting fibrosis in two MF mouse models of common MPN oncogenes Jak2 and Mpn. Mice in the first model express the constitutively active human Jak2V617F mutant under the control of the ubiquitous hematopoietic promoter vav1 and show a PV-like phenotype at 6 weeks old, progressing to fibrosis of bone marrow (BM) and spleen around 25-30 weeks. Thirty week old mice were treated with Galunisertib for 4 weeks and showed a significant decrease in fibrosis compared to control mice. Similar results were observed in 50 week old mice treated with a murine antibody against TGFβRII (IMC-TR1,LY3022859) for 4 weeks.Our second model relies on transplantation of mice with BM cells transduced with MPLW515L, a mutant mpl variant that is active in the absence of TPO. These mice rapidly develop MF, myeloproliferation and splenomegaly. Flow cytometry showed an expansion of immature and mature megakaryocytes with overexpression of TGF-β1 in MPLW515L cells. Treatment from day 12 to day 26 after transplantation reduced both white blood cell counts (a measure of myeloproliferation) and fibrosis as measured by reticulin staining. In vitro, Galunisertib counteracted the pro-fibrotic effects of TGF-ß1 on both murine and human MSC by antagonizing its stimulatory effects on collagen I and III production. Analysis of MSCs from Galunisertib-treated mice also showed reduced collagen I and III production compared to controls. In conclusion, we offer further evidence that aberrant megakaryopoiesis drives TGF-β1 overproduction in MPN by increasing megakaryocyte numbers and overexpressing TGF-β1 in mutant MKs. Galunisertib counteracts TGF-ß1 induced fibrosis in two MPN mouse-models and is a promising candidate for symptomatic treatment of myelofibrosis. Citation Format: Matthias Bartenstein, Lanzhu Yue, Wanke Zhao, Wanting Tina Ho, Cem Murdun, Adam W. Mailloux, Ling Zhang, Anjali Budhakoti, Kith Pradhan, Franck Rapaport, Huaquan Wang, Zonghong Shao, Ulrich Steidl, Ross L. Levine, Zhizhuang Joe Zhao, Amit K. Verma, Pearlie K. Epling-Burnette. TGF-ß signaling inhibition counteracts myelofibrosis in MPN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2017-954

Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells. Although significant progress has been made in treating many types of cancers during recent years, AML remains a deadly disease with survival rate lagging behind other blood cancers. A combination of toxic chemotherapies has been the standard AML treatment for more than 40 years. With intensive efforts to define the pathogenesis of AML, novel therapeutic drugs targeting key molecular defects in AML are being developed. Mutated in nearly 30% of AML, FMS-like tyrosine kinase 3 (FLT3) represents one of the most attractive targets. FLT3 mutants resulted from either internal tandem duplication (ITD) or point mutations possess enhanced kinase activity and cause constitutive activation of signaling. To date, several small molecule inhibitors of FLT3 have been developed but their clinical efficacy is limited due to a lack of potency and the generation of drug resistance. Therefore, next-generation FLT3 inhibitors overcoming these limitations are urgently in need. This review focuses on the pathological role of mutant FLT3 in the development of AML, the current status of FLT3 inhibitor development, and mechanisms underlining the development of resistance to existing FLT3 inhibitors.