Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis

Jun Ueda, Akihito Harada, Takashi Urahama, Shinichi Machida, Kazumitsu Maehara, Masashi Hada, Yoshinori Makino, Jumpei Nogami, Naoki Horikoshi, Akihisa Osakabe, Hiroyuki Taguchi, Hiroki Tanaka, Hiroaki Tachiwana, Tatsuma Yao, Minami Yamada, Takashi Iwamoto, Ayako Isotani, Masahito Ikawa, Taro Tachibana, Yuki Okada, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Kazuo Yamagata, Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis, Cell Reports, Volume 18, Issue 3, 2017, Pages 593-600, ISSN 2211-1247, https://doi.org/10.1016/j.celrep.2016.12.065

Therapeutic Advances in Immunotherapies for Hematological Malignancies

Following the success of immunotherapies such as chimeric antigen receptor transgenic T-cell (CAR-T) therapy, bispecific T-cell engager therapy, and immune checkpoint inhibitors in the treatment of hematologic malignancies, further studies are underway to improve the efficacy of these immunotherapies and to reduce the complications associated with their use in combination with other immune checkpoint inhibitors and conventional chemotherapy. Studies of novel therapeutic strategies such as bispecific (tandem or dual) CAR-T, bispecific killer cell engager, trispecific killer cell engager, and dual affinity retargeting therapies are also underway. Because of these studies and the discovery of novel immunotherapeutic target molecules, the use of immunotherapy for diseases initially thought to be less promising to treat with this treatment method, such as acute myeloid leukemia and T-cell hematologic tumors, has become a reality. Thus, in this coming era of new transplantation- and chemotherapy-free treatment strategies, it is imperative for both scientists and clinicians to understand the molecular immunity of hematologic malignancies. In this review, we focus on the remarkable development of immunotherapies that could change the prognosis of hematologic diseases. We also review the molecular mechanisms, development processes, clinical efficacies, and problems of new agents

Inhibition of the STAT5/Pim Kinase Axis Enhances Cytotoxic Effects of Proteasome Inhibitors on FLT3-ITD–Positive AML Cells by Cooperatively Inhibiting the mTORC1/4EBP1/S6K/Mcl-1 Pathway

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former conferring a poor prognosis. We have recently revealed that FLT3-ITD confers resistance to the PI3K/AKT pathway inhibitors by protecting the mTORC1/4EBP1/Mcl-1 pathway through Pim kinases induced by STAT5 activation in AML. The proteasome inhibitor bortezomib has recently been reported as a promising agent for treatment of AML. Here, we show that the proteasome inhibitor bortezomib as well as carfilzomib induces apoptosis through the intrinsic pathway more conspicuously in cells transformed by FLT3-TKD than FLT3-ITD. Mechanistically, bortezomib upregulated the stress-regulated protein REDD1 and induced downregulation of the mTORC1 pathway more distinctively in cells transformed by FLT3-TKD than FLT-ITD, while overexpression of Pim-1 partly prevented this downregulation and apoptosis in FLT3-TKD–transformed cells. Genetic enhancement of the REDD1 induction or pharmacological inhibition of STAT5, Pim kinases, mTORC1, or S6K by specific inhibitors, such as pimozide, AZD1208, PIM447, rapamycin, and PF-4708671, accelerated the downregulation of mTORC1/Mcl-1 pathway to enhance bortezomib-induced apoptosis in FLT3-ITD–expressing cells, including primary AML cells, while overexpression of Mcl-1 prevented induction of apoptosis. Thus, FLT3-ITD confers a resistance to the proteasome inhibitors on AML cells by protecting the mTORC1/Mcl-1 pathway through the STAT5/Pim axis, and inhibition of these signaling events remarkably enhances the therapeutic efficacy

FLT3-ITD Activates RSK1 to Enhance Proliferation and Survival of AML Cells by Activating mTORC1 and eIF4B Cooperatively with PIM or PI3K and by Inhibiting Bad and BIM

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously found that FLT3-ITD activates the mTORC1/S6K/4EBP1 pathway cooperatively through the STAT5/PIM and PI3K/AKT pathways to promote proliferation and survival by enhancing the eIF4F complex formation required for cap-dependent translation. Here, we show that, in contrast to BCR/ABL causing Ph-positive leukemias, FLT3-ITD distinctively activates the serine/threonine kinases RSK1/2 through activation of the MEK/ERK pathway and PDK1 to transduce signals required for FLT3-ITD-dependent, but not BCR/ABL-dependent, proliferation and survival of various cells, including MV4-11. Activation of the MEK/ERK pathway by FLT3-ITD and its negative feedback regulation by RSK were mediated by Gab2/SHP2 interaction. RSK1 phosphorylated S6RP on S235/S236, TSC2 on S1798, and eIF4B on S422 and, in cooperation with PIM, on S406, thus activating the mTORC1/S6K/4EBP1 pathway and eIF4B cooperatively with PIM. RSK1 also phosphorylated Bad on S75 and downregulated BIM-EL in cooperation with ERK. Furthermore, inhibition of RSK1 increased sensitivities to BH3 mimetics inhibiting Mcl-1 or Bcl-2 and induced activation of Bax, leading to apoptosis, as well as inhibition of proliferation synergistically with inhibition of PIM or PI3K. Thus, RSK1 represents a promising target, particularly in combination with PIM or PI3K, as well as anti-apoptotic Bcl-2 family members, for novel therapeutic strategies against therapy-resistant FLT3-ITD-positive AML

Inhibition of USP9X Downregulates JAK2-V617F and Induces Apoptosis Synergistically with BH3 Mimetics Preferentially in Ruxolitinib-Persistent JAK2-V617F-Positive Leukemic Cells

JAK2-V617F plays a key role in the pathogenesis of myeloproliferative neoplasm. However, its inhibitor ruxolitinib has shown limited clinical efficacies because of the ruxolitinib-persistent proliferation of JAK2-V617F-positive cells. We here demonstrate that the USP9X inhibitor WP1130 or EOAI3402143 (G9) inhibited proliferation and induced apoptosis more efficiently in cells dependent on JAK2-V617F than on cytokine-activated JAK2. WP1130 preferentially downregulated activated and autophosphorylated JAK2-V617F by enhancing its K63-linked polyubiquitination and inducing its aggresomal translocation to block downstream signaling. Furthermore, JAK2-V617F associated physically with USP9X in leukemic HEL cells. Induction of apoptosis by inhibition of USP9X was mediated through the intrinsic mitochondria-mediated pathway, synergistically enhanced by BH3 mimetics, prevented by overexpression of Bcl-xL, and required oxidative stress to activate stress-related MAP kinases p38 and JNK as well as DNA damage responses in HEL cells. Although autophosphorylated JAK2-V617F was resistant to WP1130 in the ruxolitinib-persistent HEL-R cells, these cells expressed Bcl-2 and Bcl-xL at lower levels and showed an increased sensitivity to WP1130 as well as BH3 mimetics as compared with ruxolitinib-naive HEL cells. Thus, USP9X represents a promising target along with anti-apoptotic Bcl-2 family members for novel therapeutic strategies against JAK2-V617F-positive myeloproliferative neoplasms, particularly under the ruxolitinib persistence conditions

Proliferation and Survival Signaling from Both Jak2-V617F and Lyn Involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 Cell Line Newly Established from Acute Myeloid Leukemia Transformed from Polycythemia Vera

<div><p>The gain of function mutation <i>JAK2-V617F</i> is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for <i>JAK2-V617F</i> from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of <i>JAK2</i> revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.</p></div

JakI-1 or dasatinib induces apoptosis involving loss of mitochondrial membrane potential and activation of caspase-3 in PVTL-1 cells.

<p>PVTL-1 cells were untreated as control (Cont.) or treated with 1 µM JakI-1 or 10 nM dasatinib, as indicated, for 48 h. Cells were then analyzed by flow cytometry for mitochondrial membrane potential with DiOC6, activation of caspase-3 with the cleavage-specific antibody, surface expression of Annexin V with anti-Annexin V, and cellular DNA content with PI, as indicated. Percentages of cells affected are indicated.</p

Jak2-V617F- and Lyn-mediated signaling events in PVTL-1 and HEL cells.

<p>A–E) PVTL-1 or HEL cells were treated with 1.5 µM JakI-1 or dasatinib at 20 nM (A, C, E) or 15 nM (B, D), as indicated, for 6 h (A, C, E) or 1 h (B, D) and lysed. Total cell lysates were subjected to Western blot analyses using indicated antibodies. Abbreviations used are: Jak2-PY, phospho-Y1007/1008-Jak2; STAT5-PY, phospho-Y694-STAT5; Lyn-PY, phospho-Y396-Lyn; 4EBP1-nonP, non-phospho-T46-4EBP1; 4EBP1-P, phospho-T37/46-4EBP1; Erk-P, phospho-T202/Y204-Erk; mTOR-P, phospho-S2448-mTOR; S6K-P, phospho-T389-p70S6K; S6RP-P, phospho-S240/244-S6RP; eIF4B-P, phospho-S422-eIF4B, GSK3-P, phospho-S21/9-GSK3α/ß. F) PVTL-1 cells were precultured with or without 5 µM SB216763 for 1 h and further cultured with or without 1.5 µM JakI-1 or 20 nM dasatinib, as indicated, for 24 h. Cells were analyzed for cellular DNA content with PI by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. G) PVTL-1 cells were precultured with or without 10 µM SB216763 for 1 h and further cultured with or without 1.5 µM JakI-1 or 20 nM dasatinib, as indicated, for 24 h. Cells were lysed and subjected to Western blot analyses using indicated antibodies. Cl. Casp-3: Cleaved Caspase-3.</p