Myeloproliferative neoplasms (MPN) are characterized by chronic proliferation of myeloid cells, extramedullary hematopoiesis and occasional leukemic transformation. Mutations in JAK2, CALR and MPL ...have been established as drivers of myeloproliferative phenotype, but their roles in disease progression with clonal expansion remain unclear. In addition, studies have shown mutations in epigenetic modifiers including TET2, DNMT3A, ASXL1 and EZH2, and aberrant expressions of microRNAs in MPN, but downstream of these changes is also largely unknown. Recently, we showed high expression of HMGA2 mRNA partly correlated with reduced microRNA let-7 in granulocytes of patients with MPN, including 100% patients with primary myelofibrosis (MF) and 20% polycythemia vera and essential thrombocythemia (Harada-Shirado et al, Brit J Haematol, 2015). In mice, loss of epigenetic modifiers such as BMI1 and EZH2, along with the Arf/Ink4a knockout (Oguro et al, J Exp Med, 2012) or the JAK2 V617F (Sashida et al, ASH, 2013), leads to overexpression of HMGA2 with accelerating MPN. We have generated transgenic (Tg) mice of Hmga2 cDNA with truncated 3'UTR (ΔHmga2) lacking binding sites of let-7 thatrepresses expression of HMGA2 (Ikeda et al, Blood, 2011). Δ Hmga2 mice overexpress HMGA2 and develop MPN-like disease, and represent a clonal advantage in competitive repopulations with serial bone marrow (BM) transplants (BMT). Here, to clarify if HMGA2 affect JAK2 V617F+ hematopoiesis, we crossed Δ Hmga2+/- mice with JAK2 V617F+/- Tg mice (Shide et al, Leukemia, 2008). Δ Hmga2-/-JAK2 V617F-/- wild type (WT), Δ Hmga2+/-JAK2 V617F-/- (Δ Hmga2 -Tg), Δ Hmga2-/-JAK2 V617F+/- (JAK2 V617F-Tg) and Δ Hmga2+/-JAK2 V617F+/- (double-Tg) mice were born at expected Mendelian ratios and we could analyze 5 - 6 of each. At 3 months old, leukocytosis, thrombocytosis, anemia and splenomegaly were most severe in double-Tg compared with JAK2 V617F-Tg or Δ Hmga2 -Tg mice. Relative to WT, peripheral leukocyte and platelet counts were nearly 16- and 4-fold higher in double-Tg, while 3- and 2-fold higher in JAK2 V617F-Tg mice, respectively. Mean spleen weights were 0.067, 0.10, 0.83 and 2.8 g in WT, Δ Hmga2 -Tg, JAK2 V617F-Tg and double-Tg mice, while BM cell counts were 2.4, 2.8, 0.4 and 1.2 x 107/femur, respectively. However, JAK2 V617F-Tg and double-Tg equally showed MF whereas no MF was detected in WT and DHmga2-Tg, suggesting that HMGA2 partly recovers cellularity in fibrotic BM. In the absence and presence of JAK2 V617F, HMGA2 augments lineage- Sca1+ Kit+ cells (WT: Δ Hmga2-Tg: JAK2 V617F-Tg: double-Tg= 0.17%: 0.19%: 0.17%: 0.27% in BM cells), endogenous erythroid colonies (1: 11: 13: 21 CFU-E/104 BM cells) and CD71+ Ter119+ erythroblasts (23%: 29%: 5.7%: 10% in BM and 2.0%: 4.4%: 7.9%: 16% in spleen cells), indicating HMGA2 contributes to expansion of hematopoietic stem/progenitor cells (HSPC) and erythroid commitment in JAK2 V617F+ hematopoiesis. Most Δ Hmga2-Tg and JAK2 V617F-Tg survived for over one year, but all double-Tg mice died within 4 months after birth due to severe splenomegaly and MF with no acute leukemia. To study the effect of HMGA2 on JAK2 V617F+ HSPC activity, we performed BMT with 0.25 x 106 Ly5.2+Δ Hmga2-Tg, JAK2 V617F-Tg or double-Tg cells with 0.75 x 106 Ly5.1+ competitor WT cells to lethally irradiated Ly5.1+ WT mice. Proportions of Ly5.2+ cells were higher in recipients of Δ Hmga2 -Tg than double-Tg cells, while JAK2 V617F-Tg cells were almost rejected at 8 weeks after BMT. To confirm role of HMGA2 without let-7 repression in JAK2 V617F+ hematopoiesis, we performed another BMT with 1 x 104 KIT+ cells of JAK2 V617F-Tg mice transduced with retroviral vector of Hmga2 with each let-7 -site-mutated full-length 3'UTR (Hmga2-m7) to sublethally irradiated WT mice. Recipients of JAK2 V617F-Tg cells with Hmga2-m7 developed MPN-like disease, whereas donor cells were rejected in recipients of JAK2 V617F cells with empty vector. In conclusion, HMGA2 may play a crucial role in hematopoiesis harboring JAK2 V617F by expanding HSPC, leading to disease progression.
No relevant conflicts of interest to declare.
Adult T‐cell leukemia/lymphoma (ATL) is an aggressive peripheral T‐cell neoplasm, and the outcome of patients with ATL after chemotherapy is poor. Allogeneic hematopoietic stem‐cell transplantation ...(allo‐HSCT) is a curative treatment modality for ATL, and four factors, namely, age > 50 years, male recipient, lack of complete remission at transplantation, and transplantation of cord blood, were previously shown to be associated with poor survival. We retrospectively analyzed the outcome of 21 patients with ATL who had undergone allo‐HSCT at our hospital during a 3‐year period. Of 21 patients, all had at least one of the above risk factors, and 18 had two or more. With a median follow‐up of 19.7 months for living patients, the 1‐ and 2‐year overall survival (OS) rates after transplantation were 34% and 27%, respectively. All relapse/progression events occurred within 1 year after allo‐HSCT, and the cumulative incidence of relapse/progression at 1 year after allo‐HSCT was 46.9%. The 100‐day and 1‐year nonrelapse mortality (NRM) rates were 19% and 42%, respectively. No significant difference in OS was observed between myeloablative and reduced‐intensity conditioning regimens. The 3‐year OS (27%) of ATL patients who received allo‐HSCT and who had at least one adverse factor was somewhat poorer than the 3‐year OS of 33% identified in a nationwide study of allo‐HSCT in ATL patients in Japan. The high relapse/progression and NRM rates are major problems to be solved to achieve better outcome.
Introduction
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T cell neoplasm that is resistant to conventional chemotherapy and carries a poor prognosis. The effect of ...mogamulizumab, an immunoglobulin (Ig) G1 monoclonal antibody targeting CCR4 for ATLL cells, was reported in a previous phase 2 study in which mogamulizumab monotherapy was evaluated in relapsed ATLL patients. The overall response rate (ORR), median progression-free survival (PFS) and median overall survival (OS) were 50%, 5.2 and 13.7 months, respectively. It was not stated whether these values were derived in the real world or in clinical practice. Here we evaluate the clinical impact of mogamulizumab treatment in CCR-4-positive aggressive ATLL patients in clinical practice.
Patients and methods
We retrospectively analyzed 101 CCR-4-positive ATLL patients who received at least one cycle of mogamulizumab infusion between March, 2012 and April, 2016 in 7 facilities in Miyazaki prefecture, an HTLV-1 endemic area in Southwestern Japan. The ORR, PFS, OS and adverse effects (AEs) were evaluated. We next compared OS in patients with at least one course of mogamulizumab therapy with that in historical control patients without mogamulizumab therapy.
Results
Of the 101 patients, 92 were evaluable for treatment response, survival and AEs. The median age was 70 years old (range; 45 to 90), and 52 patients (51%) were more than 70 years old. According to Shimoyama's criteria, 66 patients were classified as acute type, 32 as lymphoma type, and 3 as chronic type. All 3 chronic-type ATLL patients had at least one unfavorable risk factor. Of the 101 patients, 96 had refractory or relapsed ATLL when mogamulizumab treatment was started, and the prior treatments consisted of VCAP-AMP-VECP, CHOP, DeVIC or CHASE therapy, with an average of 2 courses. In the 5 remaining cases, mogamulizumab was administered as the initial therapy for ATLL. Mogamulizumab was administered as monotherapy in 87 cases (86%), and as combination therapy with other drugs in 14 cases (14%).
The ORR was 37%, including a complete remission rate of 19%. The median PFS and OS were 1.8 and 4.2 months, respectively. Among the 101 patients treated with mogamulizumab, only 26 (26%) fulfilled the inclusion criteria of the phase 2 clinical study. Among patients who met those inclusion criteria, the median PFS and OS were 6.0 and 8.4 months, respectively. The use of mogamulizumab improved OS in clinical practice. The median OS of patients receiving mogamulizumab therapy was 12 months, whereas that of patients who did not receive mogamulizumab in the historical cohort was 8.4 months. Hematologic toxicity and skin rash were the most common AEs, and both were manageable
Conclusion
Mogamulizumab therapy showed clinically meaningful activity in ATLL patients, with an acceptable toxicity in clinical practice.
No relevant conflicts of interest to declare.
MPN harbors altered hematopoietic stem cell (HSC) function, resulting in skewed hematopoiesis and extramedullary hematopoiesis with splenomegaly. Mutations such as JAK2V617F and insertion/deletion of ...CALR exon9 have been established as phenotypic drivers of MPN. In addition, mutations in epigenetic modifiers and aberrant expressions of microRNAs play a crucial role in disease progression and clonal expansion. We have shown that almost all patients with myelofibrosis (MF) highly express HMGA2 (Harada-Shirado et al, BJH, 2015) and that transgenic mice expressing HMGA2 without 3’UTR including let7 binding sites (ΔHmga2 : H) (Ikeda et al, Blood, 2011) develop mild MPN. Moreover, we (Sashida et al, JEM, 2016) and other groups (Shimizu et al, JEM, 2016; Yang et al, Blood, 2016) showed that loss of EZH2 induces endogenous expression of HMGA2 and provokes severe MF in mice carrying JAK2V617F. Thus, we hypothesized that HMGA2 plays a central role in the disease progression of MPN. To clarify this, we generated ΔHmga2/JAK2V617F transgenic mice (HJ), which developed severe leukocytosis, thrombocytosis, anemia, giant splenomegaly and shorter survival period, but did not progress fibrosis compared with transgenic mice carrying JAK2V617F alone (J) (Ueda et al, ASH, 2015). Compared with J, HJ also showed an increased bone marrow (BM) lineage-Sca1+Kit+(LSK) cells and growth advantage in competitive serial BM transplants.
Encouraged by these findings, we further investigated the mechanism that HMGA2 exacerbates disease phenotype, and elucidated up-stream and down-stream factors of HMGA2. First, we sought cause of aggressive phenotypes. In BM cells of HJ mice, STAT3 and STAT5 were drastically upregulated in both expression and phosphorylation. Despite severer anemia, formations of EPO-independent erythroid colonies and proportions of CD71+Ter119+ erythroblasts in BM were not different between HJ and J. To explain this discrepancy, we measured spontaneous apoptosis. Erythroblasts were more susceptible to apoptosis in HJ as well as aged H, compared with J, suggesting that HMGA2 contributes to apoptosis under stressed condition.
Next, we compared gene expression profiles of BM LSK cells between HJ and J by RNA sequence to seek genes altered by addition of HMGA2 expression to JAK2V617F. To clarify the role of endogenous expression of HMGA2 induced by EZH2 deletion in hematopoiesis with JAK2V617F in addition to direct effects of EZH2 deletion, we also studied RNA sequence in BM LSK cells of Ezh2-/-JAK2V617F-Tg mice (EJ). Upregulated genes were highly overlapped in between HJ and EJ. Relative to J, 200 genes including oncogenic Lmo1 were commonly upregulated, while 314 and 739 were exclusively upregulated in HJ and EJ, respectively. Thus, some of the EZH2 targets may be also targets of HMGA2. Pathway analysis revealed that chaperon-related pathways involving SCAP1 and SREBF1/2 were commonly upregulated. On the other hand, TGFB pathway was upregulated in EJ but not in HJ, and it may explain the discrepancy that addition of HMGA2 in JAK2V617F hematopoiesis did not progress fibrosis despite severe phenotype of MPN, but deletion of EZH2 exacerbated fibrosis as well as MF patients with high expression of HMGA2. To verify this finding, we measured expression of HMGA2 and its main suppressor let7, and performed target sequencing in blood samples from 16 MF patients. Because all of them highly expressed HMGA2, we also evaluated selected essential thrombocythemia (ET) patients (N=10 for HMGA2 low, 10 for HMGA2 high). While high HMGA2 expression was consistently correlated with low let-7 expression in ET, only half of MF showed low expression of let7. The other half of MF harbored mutations in components or modifiers of polycomb, including EZH2, ASXL1 and spliceosomes. These findings are compatible with the finding that loss of polycomb function upregulates HMGA2.
In fact, the genetic depletion of HMGA2 or administration of HMGA2 inhibitor neuropsin diminished phenotype of EJ mice and prolonged survival. Our findings suggest importance of HMGA2 as a therapeutic target of MPN.
Komatsu:Shire: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.
JAK2V617F (JAK2VF) is the most frequent mutation in myeloproliferative neoplasms (MPN), and its role has been demonstrated in mouse models. Actually, JAK2VF transgenic (JAK2VF Tg) mice generated by ...us induce lethal MPN (Shide et al. Leukemia 2008). Recently, mutations of epigenetic regulator such as TET2 are also frequently identified in MPN, and several TET2 knock out or knock down (TET2KD) mouse models are generated. We previously analyzed TET2KD mice (Ayu17-449) (Shide et al. Leukemia 2012). TET2KD fetal liver (FL) or bone marrow (BM) cells showed a growth advantage over Wt BM cells, with increased self-renewal capacity of hematopoietic stem cells; however TET2KD mice didn't develop MPN, and its role in MPN remained unclear. To explore the role of TET2 deficiency in MPN harboring JAK2VF, we examined the cooperative effect, using these mutant mice.
(1) Mice and collection of test cells. JAK2VF Tg mice (C57BL/6, Ly5.2) and TET2KD mice (Ayu17-449, C57BL/6, Ly5.2) were used. We crossed them, and collected JAK2Wt-TET2Wt (Wt-Wt), JAK2Wt-TET2KD (Wt-KD), JAK2VF-TET2Wt (VF-Wt), and JAK2VF-TET2KD (VF-KD) FL cells. (2) Non-competitive repopulation assay (NCRA). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) without competitor cells. Recipients were analyzed by complete blood counts, flow cytometry, colony-forming assay, colony-replating assay, pathology at 20-28 weeks post-transplantation, and overall survival. (3) Competitive repopulation assay (CRA) and serial BM transplantation (sBMT). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) with competitor Wt BM cells (Ly5.1, 5x106 cells), and sBMT was performed by 1x106 BM cells of the recipients at every 12 weeks post-transplantation. Recipients which were not selected as the donors were analyzed. (4) Analyses of adult mutant mice. Mice were bred in BDF1 background and analyzed at 20 or more weeks of age, as well as the recipients in NCRA. (5) Statistical analysis. Results were presented as means±S.D. Two-tailed Student's t-test and log-rank test were used.
In NCRA, both recipients transplanted with VF-Wt cells and VF-KD cells developed MPN with increase in WBC and Plt, decrease in Hb, fibrosis in BM and spleen, and extramedullary hematopoiesis (EMH) of lung and liver; and the latter developed more severe MPN and died earlier: VF-Wt (n=10) vs. VF-KD (n=10); WBC (x104/µl), 4.2±1.6 vs. 7.3±3.3 (p<0.05); peripheral blood (PB) myeloid cells (%), 59.6±9.7 vs. 71.9±8.2 (p<0.05); liver weight (g), 1.15±0.22 vs. 1.48±0.22 (p<0.01); spleen weight (g), 0.26±0.11 vs. 0.52±0.19 (p<0.01): VF-Wt (n=36) vs. VF-KD (n=30); mean survival time (weeks), 36 vs. 39 (p<0.05). In colony-forming assay, number of CFU-GM was more increased in VF-KD cells than VF-Wt cells: VF-Wt (n=9) vs. VF-KD (n=9); colonies/2x104 BM cells, 107±37 vs. 157±46 (p<0.05). In colony-replating assay, VF-Wt BM cells lost replating capacity by 3rd to 5th passage; VF-KD BM cells retained replating capacity beyond 5th passage: VF-Wt (n=9) vs. VF-KD (n=6); number of colonies in 4th passage, 5.9±6.8 vs. 896±613 (p<0.01). In CRA, all recipients transplanted with VF-Wt cells (n=9) or VF-KD cells (n=9) showed ≥ 70% test cell-derived PB chimerism, and developed MPN with fibrosis and EMH at 12 weeks. In 2nd BMT, 4/9 recipients transplanted with VF-Wt cells showed ≥ 35% PB chimerism at 12 weeks. Six recipients were analyzed at 12-16weeks, and no one (0/6) showed pathological findings of MPN. Whereas, 7/9 recipients transplanted with VF-KD cells showed ≥ 35% PB chimerism. Five recipients were analyzed, and 3/5 developed MPN with fibrosis and EMH: VF-Wt (n=6) vs. VF-KD (n=5); liver weight (g), 1.00±0.12 vs. 1.39±0.15 (p<0.002); spleen weight (g), 0.069±0.019 vs. 0.20±0.097 (p<0.05). In analyses of adult mutant mice, both VF-Wt mice and VF-KD mice developed MPN, and disease severities or colony-replating capacities are similar tendencies as those in transplantation model.
TET2 deficiency increases severity of MPN harboring JAK2VF. TET2 deficiency enhances disease initiating potential of JAK2VF-MPN stem cells. TET2 deficiency is considered to be critical for both onset and progression of MPN harboring JAK2V617F.
No relevant conflicts of interest to declare.
Summary
The acquired JAK2 V617F mutation is observed in the majority of patients with BCR‐ABL1 negative chronic myeloproliferative neoplasms (MPN). BCR‐ABL1 negative MPN displays myeloproliferation ...with an elevated leucocyte alkaline phosphatase (LAP) activity, a neutrophil activation marker. We tried to separate the downstream signalling of JAK2 V617F to stimulate myeloproliferation and LAP activity. NB4, a myeloid lineage cell line, was transduced with Jak2 V617F mutation or wild‐type Jak2. We found that Jak2 V617F mutation, but not wild‐type Jak2 enhanced LAP expression in NB4‐derived neutrophils and proliferation of NB4 cells. JAK2 V617F induces constitutive phosphorylation of STAT3 and STAT5, and uses signalling targets such as Ras/MEK/ERK and PI3K/Akt pathways. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, and STAT3 or STAT5 siRNAs, JAK2 V617F was found to specifically use the STAT3 pathway to enhance LAP expression, while STAT5, Ras/MEK/ERK and PI3K/Akt, but not STAT3 pathways, were able to stimulate cell proliferation. These data strongly suggest that JAK2 V617F uses distinct signalling pathways to induce typical pathological features of MPN, such as high LAP activity and enhanced cell proliferation.
► Tyk2 interacts with the proapoptotic protein Siva-1 via its N-terminus region. ► Tyk2 phosphorylates Siva-1, which has little effect on Siva-1-induced apoptosis. ► Tyk2 augments the apoptotic ...function of Siva-1 through the Tyk2-Siva-1 interaction.
Siva-1 is a molecule that has the potential to induce both extrinsic (receptor-mediated) and intrinsic (non-receptor-mediated) apoptosis. Siva-1 binds to CD27, a member of the tumor necrosis factor receptor (TNFR) family, Abl-related gene (ARG), and BCL-X
L, and these partner molecules reportedly enhance the apoptotic properties of Siva-1. In this study, we show that Siva-1 also interacts with a member of the Jak family protein kinases, tyrosine kinase 2 (Tyk2). Siva-1 bound to Tyk2 via its N-terminal region, and Tyk2 phosphorylated Siva-1 at tyrosines 53 and 162. In murine pro-B cells, Ba/F3 cells, expression of Tyk2 augmented Siva-1-induced apoptosis. This augmentation of Siva-1-induced apoptosis was retained regardless of the phosphorylation of Siva-1, but was almost completely prevented by the abrogation of the Tyk2-Siva-1 association. These findings indicate that the interaction between Siva-1 and Tyk2 directly augments the apoptotic activity of Siva-1. Our novel observations suggest that Siva-1 forms a functional complex with Tyk2 and participates in the transduction of signals that inhibit B lymphocyte growth.
Objective
The efficacy of mogamulizumab in adult T‐cell leukemia/lymphoma (ATLL) was reported in a previous phase 2 study. Compared with patients in clinical trials, however, most patients in ...real‐life settings have demonstrated worse outcomes.
Method
We retrospectively analyzed 96 patients with relapsed/refractory ATLL who received mogamulizumab treatment.
Results
Relapsed/refractory ATLL patients with a median age of 70 years received a median of five courses of mogamulizumab. Hematologic toxicity and skin rash were the most common adverse events, and both were manageable. Of 96 patients, 87 were evaluable for efficacy. The overall response rate was 36%, and the median progression‐free survival (PFS) and overall survival (OS) from the start of mogamulizumab therapy were 1.8 and 4.0 months, respectively. Of the original 96 patients, only 25 fulfilled the inclusion criteria of the phase 2 study. Those who met the criteria demonstrated longer median PFS and OS durations of 2.7 and 8.5 months, respectively. The median OS from diagnosis in relapsed/refractory ATLL patients receiving mogamulizumab was 12 months, longer than the 5.8 months in a historical cohort without mogamulizumab.
Conclusion
In clinical practice, mogamulizumab exhibited antitumor activity in patients with relapsed/refractory ATLL, with an acceptable toxicity profile. Mogamulizumab therapy improved the OS of ATLL patients.
PDF
