Glycogen synthase kinase 3beta missplicing contributes to leukemia stem cell generation.

Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of beta-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving beta-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus are enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/beta-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3beta, axin 1, beta-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3beta kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3beta have enhanced beta-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3beta reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3beta in GMP LSC, enabling unphosphorylated beta-catenin to participate in LSC self-renewal. Missplicing of GSK3beta represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.

SM04755, a small-molecule inhibitor of the Wnt pathway, as a potential topical treatment for tendinopathy.

The Wnt pathway is upregulated in tendinopathy, affecting inflammation and tenocyte differentiation. Given its potential role in tendinopathy, this signaling pathway may be a relevant target for treatment. The current study examined the therapeutic potential of SM04755, a topical, small-molecule Wnt pathway inhibitor, for the treatment of tendinopathy using in vitro assays and animal models. In vitro, SM04755 decreased Wnt pathway activity, induced tenocyte differentiation, and inhibited catabolic enzymes and pro-inflammatory cytokines in human mesenchymal stem cells, rat tendon-derived stem cells, and human peripheral blood mononuclear cells. Evaluation of the mechanism of action of SM04755 by biochemical profiling and computational modeling identified CDC-like kinase 2 (CLK2) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as molecular targets. CLK and DYRK1A inhibition by siRNA knockdown or pharmacological inhibition induced tenocyte differentiation and reduced tenocyte catabolism. In vivo, topically applied SM04755 showed therapeutically relevant exposure in tendons with low systemic exposure and no detectable toxicity in rats. Moreover, SM04755 showed reduced tendon inflammation and evidence of tendon regeneration, decreased pain, and improved weight-bearing function in rat collagenase-induced tendinopathy models compared with vehicle control. Together, these data demonstrate that CLK2 and DYRK1A inhibition by SM04755 resulted in Wnt pathway inhibition, enhanced tenocyte differentiation and protection, and reduced inflammation. SM04755 has the potential to benefit symptoms and modify disease processes in tendinopathy.

The CLK inhibitor SM08502 induces anti-tumor activity and reduces Wnt pathway gene expression in gastrointestinal cancer models.

The Wnt/ß-catenin signaling pathway is aberrantly activated in colorectal (CRC) and many other cancers, and novel strategies for effectively targeting it may be needed due to its complexity. In this report, SM08502, a novel small molecule in clinical development for the treatment of solid tumors, was shown to reduce Wnt pathway signaling and gene expression through potent inhibition of CDC-like kinase (CLK) activity. SM08502 inhibited serine and arginine rich splicing factor (SRSF) phosphorylation and disrupted spliceosome activity, which was associated with inhibition of Wnt pathway-related gene and protein expression. Additionally, SM08502 induced the generation of splicing variants of Wnt pathway genes, suggesting that its mechanism for inhibition of gene expression includes effects on alternative splicing. Orally administered SM08502 significantly inhibited growth of gastrointestinal tumors and decreased SRSF phosphorylation and Wnt pathway gene expression in xenograft mouse models. These data implicate CLKs in the regulation of Wnt signaling and represent a novel strategy for inhibiting Wnt pathway gene expression in cancers. SM08502 is a first-in-class CLK inhibitor being investigated in a Phase 1 clinical trial for subjects with advanced solid tumors (NCT03355066).

Thrombopoietin regulates c-Myb expression by modulating micro RNA 150 expression.

OBJECTIVE: Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytes and their progenitors. Thrombopoietin induces these same effects, which lead us to hypothesize that the hormone acts through modulation of c-Myb expression, as c-Myb levels falls during thrombopoietin-induced megakaryocyte (MK) maturation. Micro RNAs (miRs) downregulate gene expression by binding to the 3' untranslated region (UTR) of specific messenger RNAs (mRNAs); we noted that the 3'UTR of c-Myb contains four miR-150 binding sites. MATERIALS AND METHODS: We used quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene analyses to assess the response of c-Myb to thrombopoietin stimulation and to gain of and loss of miR-150 expression. RESULTS: We found that thrombopoietin reduced c-Myb mRNA and protein levels within 7 hours in megakaryocytes and UT7/thrombopoietin (TPO) cells. Using a reporter gene containing the c-Myb 3'UTR region, including its four miR150 binding sites, we found that expression of miR150 reduced luciferase expression to 50% of baseline at 24 hours and to 25% at 48 hours in UT7/TPO cells. Quantitative polymerase chain reaction and Western blotting also revealed that miR-150 reduced endogenous c-Myb mRNA and protein to 50% in UT7/TPO cells, and to 65% in mature megakaryocytes. Converse experiments utilizing anti-miR150 increased luciferase activity twofold over control anti-miR. Finally, TPO increased miR150 expression 1.8-fold within 24 hours and 3.4-fold within 48 hours. CONCLUSIONS: These findings establish that miR150 downmodulates c-Myb levels, and because TPO affects miR150 expression, our results indicate that, in addition to affecting MK progenitor cell growth, TPO downmodulates c-Myb expression through induction of miR-150.

Thrombopoietin (TPO) induces c-myc expression through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR in TPO-dependent cell lines and primary megakaryocytes.

Thrombopoietin (TPO) and its receptor (c-Mpl) are the major regulators of megakaryocyte and platelet production and serve a critical and non-redundant role in hematopoietic stem cell (HSC) biology. TPO signals through the Jak-STAT, Ras-Raf-MAPK, and PI3K pathways, and promotes survival, proliferation, and polyploidization in megakaryocytes. The proto-oncogene c-myc also plays an important role in many of these same processes. In this work we studied the regulated expression of c-myc in megakaryocytic cell lines and primary cells by quantitative real-time RT-PCR. We found that TPO induced expression of c-myc in 1 h in both hematopoietic cell lines (UT-7 and BaF3/Mpl) and mature murine megakaryocytes. The TPO-induced expression of c-myc was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting that TPO stimulated c-myc expression through a PI3K-dependent pathway. Of interest, our study showed that overexpression of active Akt did not rescue the effect of PI3K blockade on c-myc expression, rather, enhanced it. In addition, inhibitors of protein kinase C (PKC)zeta and the target of rapamycin (mTOR) also failed to affect c-myc mRNA expression, while c-myc mRNA expression was reduced by inhibition of the mitogen activated protein kinase (MAPK) pathway. Therefore, we conclude that TPO stimulates c-myc expression in primary megakaryocytes through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR.

Selective inhibition of JAK2-driven erythroid differentiation of polycythemia vera progenitors.

Polycythemia Vera (PV) is a myeloproliferative disorder (MPD) that is commonly characterized by mutant JAK2 (JAK2V617F) signaling, erythrocyte overproduction, and a propensity for thrombosis, progression to myelofibrosis, or acute leukemia. In this study, JAK2V617F expression by human hematopoietic progenitors promoted erythroid colony formation and erythroid engraftment in a bioluminescent xenogeneic immunocompromised mouse transplantation model. A selective JAK2 inhibitor, TG101348 (300 nM), significantly inhibited JAK2V617F+ progenitor-derived colony formation as well as engraftment (120 mg/kg) in xenogeneic transplantation studies. TG101348 treatment decreased GATA-1 expression, which is associated with erythroid-skewing of JAK2V617F+ progenitor differentiation, and inhibited STAT5 as well as GATA S310 phosphorylation. Thus, TG101348 may be an effective inhibitor of JAK2V617F+ MPDs in clinical trials.

Genetic influence of CCR5, CCR2, and SDF1 variants on human immunodeficiency virus 1 (HIV-1)-related disease progression and neurological impairment, in children with symptomatic HIV-1 infection.

The role that host genetics plays in the modification of the rate of human immunodeficiency virus 1 (HIV-1)-related disease progression was evaluated in a seroprevalent cohort of 1049 children with symptomatic HIV-1 infection who participated in 2 clinical trials in the United States. Variants including CCR2-V64I, CCR5-wt/Delta32, CCR5-59029-G/A, CCR5-59353-T/C, CCR5-59356-C/T, and SDF1-3'-G/A were identified by polymerase chain-reaction genotyping. Children with the CCR5-wt/Delta32 genotype experienced significantly delayed disease progression, including less neurocognitive impairment. In the CCR5-wt/wt group, the most rapid disease progression was in those with the CCR5-59029-A/A genotype, which was present in 23% of the children. Although the SDF1-3'-A/A variant was associated with more-rapid disease progression, it occurred in <2% of the children studied. Modest or little impact was associated with the CCR5-59353, CCR5-59356, or CCR2 genotypes. Thus, in children with the CCR5-wt/wt genotype, variants at CCR5-59029 have the broadest impact on disease progression. These data suggest that, in children, host genetics plays an important role in HIV-1-related disease progression and neurological impairment.

Prevalence of chemokine and chemokine receptor polymorphisms in seroprevalent children with symptomatic HIV-1 infection in the United States.

Several chemokines and chemokine receptors are involved in HIV-1 infection, disease progression, and transmission. We studied the prevalence of genetic variations in CCR2, SDF1, and the CCR5 gene and its promoter region at positions 59029, 59353, and 59356 in a seroprevalent cohort of 1057 children with symptomatic HIV-1 infection in the United States. The percentage of children with the CCR5-wt/Delta32 genotype was significantly higher for white, non-Hispanic children (15%) than for Hispanic (6%) or black, non-Hispanic children (4%). For the CCR5-59029-G/A, CCR5-59353-T/C, and CCR5-59356-C/T polymorphisms, there were significant or marginally significant differences in genotype frequencies across race/ethnicity groups. For the CCR2-wt/64I polymorphism, both black, non-Hispanic and Hispanic children had a higher frequency of the CCR2-wt/64I genotype (24% and 21%, respectively) and CCR2-64I/64I genotype (4% and 3%, respectively) than white, non-Hispanic children (14% and 2%, respectively). For the SDF1-3'-G/A polymorphism, black, non-Hispanic children had a lower combined frequency of the SDF1-3'-G/A and SDF1-3'-A/A genotypes (15%) than did Hispanic children (33%) and white, non-Hispanic children (37%). These analyses show that the distribution of chemokine receptor and chemokine genetic polymorphisms varies significantly across race/ethnicity subgroups of HIV-1-infected children in the United States.