Objective and Method
Adult T‐cell leukemia/lymphoma (ATL) is an aggressive peripheral T‐cell lymphoma with poor prognosis. We retrospectively reviewed the medical records of 312 patients with ...aggressive ATL and analyzed the effect of chemotherapy dose intensity on prognosis in clinical practice.
Result
As first‐line therapy, 62 patients underwent best supportive care (BSC) or single‐agent chemotherapy, and 235 underwent intensive chemotherapy. The median survival time (MST) was 0.58 years in the 312 total patients, and 0.13 years and 0.75 years in the BSC/single‐agent chemotherapy group and intensive chemotherapy group, respectively. The median average relative dose intensity (ARDI) of patients who received intensive chemotherapy was 60%. We divided patients into 3 groups according to ARDI. Those in the top tertile of ARDI (ARDI ≥ 75%, n = 82) had better overall survival compared with those in the intermediate tertile (45% ≤ ARDI < 75%, n = 79) (P < .0001), with MSTs of 4.69 and 0.75 years, respectively. The occurrence of organ dysfunction and infectious complications was comparable between the two ARDI groups.
Conclusion
Higher ARDI improves prognosis in patients with aggressive ATL in clinical practice.
Calreticulin (CALR) exon 9 mutations were reported in about two-thirds of JAK2 or MPL mutation negative ET and PMF patients. The mutations cause frameshifts that result in proteins with novel ...C-terminus.Retrovirus-mediated gene transfer into cell lines and mouse bone marrow (BM) cells is a common technique, but the expression level is very high compared to the physiological expression.We investigated the effects of physiological expression of mutant CALR using CRISPR/Cas9 gene editing techniques for cell lines, and as for the mouse model, we generated a transgenic mice (TG) expressing human CALR del52 mutant.
We used two human cell lines expressing MPL: human acute megakaryoblastic leukemia cell line CMK11-5 which expressed endogenous MPL, and F-36P-MPL cell line which was generated by introducing MPL to GM-CSF-dependent erythroleukemia cell line F-36P. Plasmids coexpressing hCas9 and single-guide RNA were prepared by ligating oligonucleotides (5'-CACCGACAAGAAACGCAAAGAGGAGG-3', 5'-AAACCCTCCTCTTTGCGTTTCTTGTC-3') for the target sequence of human CALR exon 9 into pX330. The plasmids were introduced with a electroporator to each of the cell lines. After limiting dilution cloning, we identified cell lines which have indel mutation at the target site.
We produced two types of CMK11-5 subline knocked in a CALR mutation, namely CALR del25 CMK cells and CALR del25/del17 CMK cells, respectively. The former lacks 25 bases in one CALR allele, causing a frameshift that results in a protein resembling human CALR mutant, while the latter lacks an additional 17 bases in another allele in CALR exon 9 and induces a frameshift that causes a deletion in CALR exon 9. Both kinds of CALR mutant CMK11-5 cells showed increased cell proliferation compared to WT cells. We also produced one type of F-36P-MPL subline, CALR del1/ins1 F-36P-MPL cells which had 1 base deletion in one CALR allele resembling human mutation and 1 base insertion in another allele. Though the growth of this subline in the presence of GM-CSF was comparable to WT cells, it showed GM-CSF independent autonomous cell growth.
We generated TG mice expressing human CALR del52 mutant driven by the murine H2Kb promoter. We compared the expression level of human CALR mRNA in TG BM cells with the expression of endogenous WT CALR in human cell lines (CMK11-5, F-36P-MPL, CHRF288) using Rn18s as an endogenous control. The expression of human CALR in TG BM was approximately 0.6 times that of endogenous WT CALR in human cell lines, and the physiological expression level was obtained.
They exhibited thrombocytosis, with platelet (PLT) counts as high as 2,000 x 109/L. Leukocyte number and the proportion of granulocytes and T and B lymphocytes, were comparable to WT mice. CALR mutation had no impact on Hb level or spleen weight. There was a striking difference in the number of megakaryocytes (Mgks), which was 2-fold higher in BM from TG mice than in WT mice. The TG Mgks were also more mature, with larger diameter, and contained higher number of alpha-granules compared to WT cells. In one year of observation, there is no fibrosis in BM. These observations showed that TG developed human ET-like disease. The survival of TG mice was comparable to that of WT mice. The disease phenotype was transplantable into WT recipient mice. To characterize in detail the impact of MPNs induced by the CALR del52 mutant, we evaluated the frequencies of HSCs and progenitors in BM. The frequency of both LT-HSC and ST-HSC in BM was higher inTG mice compared to WT mice. The frequencies of progenitors (CMP, MEP, and MKP) were also greater in BM from TG mice than from WT mice. However, BM cells did not have enhanced replating capacity. We next examined whether or not ruxolitinib (RUX) treatment ameliorated thrombocytosis in TG mice. Either 90 mg/kg bid of RUX or vehicle was administrated to TG mice for 4 weeks.TG mice treated with vehicle showed a mean 16% increase in PLT count during the treatment period, probably due to the disease progression. RUX treatment attenuated the increase in the number of PLTs in TG mice by a mean of 22%, but the overall count was still higher than that in WT mice. BM sections showed that RUX reduced the Mgks number in TG.
In summary, physiological expression of CALR mutant increases cell growth and cytokine independency in human cell lines expressing MPL, and develops ET in mice. RUX therapy attenuated the increased numbers of peripheral blood PLTs and BM Mgks, and ameliorated CALR mutation-induced ET.
No relevant conflicts of interest to declare.
An acquired JAK2 V617F mutation is found in most patients with polycythemia vera (PV), and about half of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). Mice transplanted ...with bone marrow cells in which JAK2 V617F was retrovirally expressed developed PV-like features, but not ET or PMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated two lines of JAK2 V617F transgenic mice. One line showed granulocytosis after 4 months of age. Among 43 mice, 8 (19%) showed polycythemia and 15 (35%) showed thrombocythemia. The second line showed extreme leukocytosis and thromobocytosis. They showed anemia that means Hb value from 9 to 10 g per 100 ml when 1 month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly was observed. The expression of JAK2 V617F mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines, respectively. In vitro analysis of bone marrow cells from both lines showed constitutive activation of ERK1/2, STAT5 and AKT, and augmentation of their phosphorylations by cytokine stimulation. We conclude that in vivo expression of JAK2 V617F results in ET-, PMF- and PV-like disease.
Mutations in JAK2, MPL and CALR are regarded as driver mutations, and are mutually exclusively detected in more than 90% of myeloproliferative neoplasms (MPNs). In addition, mutations in epigenetic ...regulator genes such as TET2 or DNMT3A are detected in MPNs. Although the roles of mutations in epigenetic regulator genes were clarified in normal hematopoiesis, their roles have remained unclear in malignant hematopoiesis of MPNs. We analyzed three lines of mutant mice: mice with JAK2V617F, a representative of driver gene mutations; mice with loss of TET2, a representative of epigenetic abnormalities; and mice with both. We thereby clarified two roles of loss of TET2 in malignant hematopoiesis of JAK2-mutated MPNs: one is "disease initiator and sustainer" via reinforcing the function of JAK2-mutated hematopoietic stem cells, and the other is "disease accelerator". New strategies in risk assessment or treatment are required, considering not only single but also multiple mutations.
Myeloproliferative neoplasms are frequently complicated by thrombosis and bleeding. Therefore, not only their primary prevention, but also the management of special clinical issues is important. ...These issues include venous thromboembolic disorders such as splanchnic venous thromboembolism, as well as major bleeding, surgery, and pregnancy. As for the primary prevention of thrombosis, it has been proposed that low-risk essential thrombocythemia with newly reported risk factors (cardiovascular risks or JAK2V617F) might be included among the factors meriting prevention. As for management of the aforementioned special clinical issues, an expert consensus has been established, wherein the recommended treatment strategies are described. In Japan as well, clinical practice based on this consensus would be preferable.
In primary myelofibrosis patients, somatic mutations such as JAK2V617F(JAKVF) and MPLW515 that activate JAK-STAT signaling are often seen. Small-molecule JAK2 inhibitors are effective for ...organomegaly and constitutional symptoms, but the drugs have little effect on BM fibrosis. To clarify the mechanism by which MPN cells with JAK2 mutations cause BM fibrosis, we compared the gene expression patterns of Lin−Sca1+ BM cells in JAK2VF transgenic mice (JAK2VF-TG), which develop myelofibrosis (MF), with that in WT mice. We found that TGFb1 and HOXB4, the target genes of transcription factor USF1 were highly expressed. TGFβ1, which is secreted by hematopoietic cells, is essential for fibrotic development in a murine model of MF (Chagraoui et al. Blood 2002), and increased expression of HOXB4 enhances human megakaryocytic development (Zhong et al. BBRC 2010).
To investigate the mechanism of the high expression of these genes downstream of JAK2 signaling, USF1 and a cytokine receptor gene (MPL, EPOR or CSF3R) were co-transfected into 293T cells along with either a TGF-β1/HOXB4 promoter-driven or a STAT5 response element-driven luciferase reporter. Stimulation of MPL with TPO enhanced USF1 transcriptional activity about 3 fold, but stimulation of EPOR with EPO or of CSF3R with G-CSF did not change this activity. However, stimulation with any of the 3 types of cytokines enhanced STAT5 transcriptional activity. JAK2VF upregulated USF1 and STAT5 much more highly than JAK2WT without TPO stimulation. This USF1 upregulation specifically to TPO/MPL signaling was suppressed by a dominant negative mutant of USF1, JAK2 inhibitors (AG490, NS-018) or MEK inhibitors (U0126, PD325901). Inhibition of PI3K or p38MAPK did not affect the USF1 activation. Co-treatment with JAK2 and MEK inhibitors showed a synergistic effect in blocking both USF1 upregulation and STAT5 activation induced by JAK2VF.
Next, we tested the MEK inhibitor, PD325901, in combination with the JAK2 inhibitor, NS-018, in the JAK2VF-TG mice. After disease was established 12 weeks after birth, JAK2VF-TG mice were divided into the following 4 groups: vehicle control; PD325901 monotherapy; NS-018 monotherapy; and combined therapy. PD325901 (5 mg/kg) and NS-018 (50 mg/kg) were orally administered once and twice daily, respectively. After 12 weeks of treatment, we evaluated the effect on BM fibrosis. The grading of MF in each group (n = 5-6) was as follows: vehicle control (MF-0: 0/6, MF-1 or 2: 6/6); PD325901 monotherapy (MF-0: 4/5, MF-1 or 2: 1/5); NS-018 monotherapy (MF-0: 0/6, MF-1 or 2: 6/6); and combined therapy (MF-0: 3/6, MF-1 or 2: 3/6). In the 2 groups treated with PD325901, 50~80% of mice showed MF-0. In contrast, in vehicle-treated or NS-018 monotherapy groups, all mice showed MF-1 or 2. Consistent with the MF grading, BM cellularity was significantly increased in the PD325901 monotherapy or combined therapy groups compared with the vehicle-treated group. A significant reduction was seen in the plasma TGFβ1 concentration in the PD325901 monotherapy and combined therapy groups compared with the vehicle-treated group (9.7 ng/ml, 8.1 ng/ml vs. 18.2 ng/ml, respectively). The TGFβ1 concentration in the extracellular fluid of BM (Wagner et al blood 2007) was also significantly reduced (5.6 ng/ml, 6.8 ng/ml vs. 9.1 ng/ml, respectively). BM cellularity and the TGFβ1 concentration in the NS-018 monotherapy group were comparable to those in the vehicle-treated group. Interestingly, megakaryocytes in the PD325901 monotherapy and combined therapy groups were decreased in number and were smaller than those in the vehicle-treated or NS-018 monotherapy groups. Regarding the effect on splenomegaly, spleen weight was significantly reduced in the NS-018 monotherapy and combined therapy groups compared with the vehicle-treated group (0.83 g, 0.69 g vs. 1.18 g, respectively). PD325901 monotherapy had little effect on splenomegaly.
It is known that MEK-ERK1/2 pathway is critical in normal megakaryocyte development. In vitro data suggest that JAK2VF activates this pathway downstream of MPL and may contribute to TGFβ1 overproduction and dysmegakaryopoiesis, causing BM fibrosis via transcriptional enhancement of USF1. In vivo data suggest that MEK inhibition has the potential to improve dysmegakaryopoiesis and BM fibrosis. The combined therapy of JAK2 inhibitors with MEK inhibitors might be a promising therapy for improving both splenomegaly and BM fibrosis.
No relevant conflicts of interest to declare.
A single somatic mutation, V617F, in Janus kinase 2 (JAK2) is one of the causes of myeloproliferative neoplasms (MPN), including primary myelofibrosis, and the mutant kinase JAK2V617F is a ...therapeutic target in MPN. However, inhibition of wild-type JAK2 (JAK2WT) can decrease the red blood cell (RBC) or platelet count. Therefore, a JAK2 inhibitor that produces a smaller reduction in the RBC and platelet counts in the therapeutic window would have clinical benefit.
NS-018 is a potent and selective inhibitor of JAK2 and Src-family kinases which is currently in an early-phase clinical trial for MPN. To compare the inhibitory effect of NS-018 on JAK2WT and JAK2V617F in the cell, we assessed the antiproliferative activity of NS-018 against Ba/F3 cells expressing murine JAK2WT or JAK2V617F. NS-018 suppressed the growth of Ba/F3-JAK2V617F cells with an IC50 value of 470 nM, whereas it suppressed the growth of Ba/F3-JAK2WT cells stimulated with IL-3 with an IC50value of 2000 nM. Thus, NS-018 showed 4.3-fold selectivity for Ba/F3-JAK2V617F over Ba/F3-JAK2WT cells (V617F/WT ratio). Other JAK2 inhibitors also showed selectivity for Ba/F3-JAK2V617F over Ba/F3-JAK2WT cells, though their selectivity was lower. For example, INCB018424 (ruxolitinib) and TG101348 showed V617F/WT ratios of 2.0 and 1.5, respectively. Among the eight JAK2 inhibitors tested, NS-018 showed the highest selectivity for JAK2V617F cells. NS-018 also inhibited erythroid colony formation in JAK2V617F transgenic mice at significantly lower concentrations than in wild-type mice.
To assess the ability of NS-018 to selectively inhibit JAK2V617F-harboring cells in vivo, we established a JAK2V617F bone marrow transplantation (BMT) mouse model. NS-018 was administered by oral gavage twice a day for 40 days at a dose of 50 mg/kg. When assessment was carried out 50 days after the start of the study, NS-018 was found to have significantly prolonged the survival of JAK2V617F BMT mice, decreased their splenomegaly and restored their disrupted splenic architecture. NS-018 also partially suppressed bone marrow fibrosis in JAK2V617F BMT mice. All vehicle-treated mice that had survived to the study endpoint had mild-to-moderate reticulin fibrosis, whereas all mice treated with NS-018 had slight-to-little reticulin fibrosis, except for one mouse with mild fibrosis. Although vehicle-treated JAK2V617F BMT mice showed marked leukocytosis, NS-018 treatment achieved a 95% suppression of this increase. In spite of the marked effects of NS-018 in JAK2V617F BMT mice described above, NS-018 treatment had not decreased the RBC or reticulocyte count after 50 days of administration. JAK2V617F BMT mice showed a 78% decrease in the platelet count compared with control mice, and NS-018 treatment did not further decrease the count.
To better understand the ability of NS-018 to preferentially inhibit the mutated form of JAK2, we explored the X-ray co-crystal structure of NS-018 bound to activated JAK2 and focused on the flipped carbonyl group of Gly933, which is located immediately N-terminal to the DFG (Asp-Phe-Gly) motif in the activation loop of JAK2. We identified two kinds of hydrogen-bonding interactions between NS-018 and the carbonyl group of Gly993: water-mediated hydrogen bonding involving a nitrogen atom of NS-018 and a CH•••O hydrogen bond involving an aromatic CH of NS-018. The unique mode of binding of NS-018 to activated JAK2 provides a plausible explanation for its JAK2V617F selectivity.
In summary, NS-018 preferentially inhibited the growth of JAK2V617F-harboring cells over JAK2WT-harboring cells. NS-018 was also effective against leukocytosis, splenomegaly, and bone marrow fibrosis, and prolonged survival in JAK2V617F BMT mice with no reduction in the RBC or platelet counts. These characteristics of NS-018 may be explained at least in part by its unique mode of binding to the activated form of JAK2. NS-018 may have therapeutic benefit for MPN patients in virtue of its simultaneous satisfaction of the two requirements of efficacy and reduced hematologic adverse effects.
Nakaya:Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Niwa:Nippon Shinyaku: Employment. Horio:Nippon Shinyaku: Employment.
Polycomb group proteins are transcriptional repressors that epigenetically regulate transcription via histone modifications. There are two major polycomb-complexes, the Polycomb Repressive Complexes ...(PRC1 and PRC2). PRC2 contains SUZ12, EED, and EZH2 that catalyze the trimethylation of histone H3 at lysine 27 (H3K27me3) and silence target-genes expression. EZH2 is generally thought to act as an oncogene in lymphoma by silencing tumor suppressor genes through H3K27me3 modifications. However, loss-of-function mutations of EZH2 have been found in myeloid malignancies such as MDS and MPN including primary myelofibrosis (PMF). In a recent study, EZH2 mutations were independently associated with shorter survival in PMF patients, suggesting that EZH2 functions as a tumor suppressor in PMF.
Although JAK2V617F mutant is found in approximately 50% of PMF patients, it remains obscure whether the presence of JAK2V617F mutant predicts survival of PMF patients, and the functional contribution of JAK2V617F to the development of PMF has not been fully delineated. JAK2 has been shown to directly phosphorylate H3Y41 (H3Y41p) and reduce HP1a binding, thereby activating expression of target genes. However, it is unknown how JAK2V617F epigenetically alter expression of target genes in the development of PMF. Given that JAK2V617F mutation is significantly associated with EZH2 mutations in PMF patients, in order to understand how EZH2 mutations contribute to the pathogenesis of JAK2V617F-positive PMF, we generated a novel mouse model of PMF utilizing H2K-JAK2V617F transgenic mice and Ezh2 conditional knockout mice.
We first harvested 5x106 bone marrow cells from tamoxifen-inducible Cre-ERT;Ezh2wild/wild (WT), Cre-ERT;Ezh2flox/flox (Ezh2 cKO), JAK2V617F TG/Cre-ERT;Ezh2wild/wild (JAK2 TG) and JAK2V617F TG/Cre-ERT;Ezh2flox/flox (JAK2 TG/Ezh2 cKO) mice, and then transplanted into lethally irradiated recipient mice. At 4 weeks post transplantation, we deleted Ezh2 via administration of tamoxifen, and observed disease progression until 9 months post transplantation. WT and Ezh2 cKO mice did not develop myeloid malignancies. While all 11 JAK2 TG mice died due to PMF-like disease after a long latency as previously reported, 10 out of 10 JAK2 TG/Ezh2 cKO mice immediately developed PMF and died by approximately 50 days post-deletion of Ezh2. JAK2 TG/Ezh2 cKO mice showed a significantly shorter median survival than did JAK2 TG mice (36.5 days versus 245 days, p<0.01). In the peripheral blood, moribund JAK2 TG/Ezh2 cKO mice showed increased mature neutrophils, severe anemia, and thrombocytopenia, compared to WT or JAK2 TG mice at 2 months post transplantation. At the time of sacrifice, JAK2 TG/Ezh2 cKO mice showed a significant hypoplastic bone marrow without an increased myeloblast cells, but also had a marked splenomegaly due to infiltration of myeloid cells compared to JAK2 TG mice. In addition, JAK2 TG/Ezh2 cKO mice showed a severe myelofibrosis in both bone marrow and spleen, indicating that Ezh2 loss obviously promotes JAK2 V617F-driven PMF in vivo.
To understand a molecular mechanism how Ezh2 functions as a tumor suppressor for PMF, we performed gene expression analysis in Lin-Sca1+c-Kit+ (LSK) cells. While Ezh2 cKO LSKs and JAK2 TG LSKs showed up-regulation (>2-fold) of 1044 and 861 genes, respectively, JAK2 TG/Ezh2 cKO LSKs showed up-regulation (>2-fold) of more genes (1306), compared to WT LSKs. As expected, H3Y41p and H3K27me3 target genes were significantly upregulated in JAK2 TG/Ezh2 cKO LSKs, whereas H3K27me3 targets were significantly repressed in JAK2 TG LSKs, consistent with the tumor suppressor role of Ezh2 in PMF. We are now working to understand how dysregulated genes are involved in the progression of JAK2V617F-induced PMF after deletion of Ezh2. In conclusion, we have successfully established the progressive PMF in mice reconstituted with Ezh2 null cells expressing JAK2V617F mutant, and demonstrated that Ezh2 functions as a tumor suppressor in this context. This model can be utilized for innovating new therapies for PMF.
No relevant conflicts of interest to declare.
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