Clinical factors for central nervous system progression and survival in primary vitreoretinal lymphoma.

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of malignant lymphoma with a poor prognosis because of high frequency of central nervous system (CNS) progression. Identification of factors associated with CNS progression is essential to improve the prognosis of patients with PVRL. We conducted a retrospective study of 54 patients diagnosed with PVRL and treated at our hospital to identify factors associated with CNS progression and prognosis. All patients were treated with intravitreal methotrexate (MTX) injections in the affected eyes until lesion resolution. Twenty-four patients were treated with systemic administration of high-dose MTX (systemic HD-MTX) every other week for a total of five cycles following intravitreal MTX injection. Of 24 patients, 20 completed five cycles of systemic HD-MTX. The 5-year cumulative incidence of CNS progression and overall survival (OS) rate were 78.0% and 69.0% respectively. By univariate and multivariate analyses, bilateral disease and the detection of B-cell clonality confirmed by flow cytometric analysis were risk factors associated with CNS progression. Moreover, systemic HD-MTX completion reduced the risk of CNS progression and was identified as a factor affecting OS. In this study, factors for CNS progression identified may potentially contribute to the optimized therapeutic stratification to improve the survival of patients with PVRL.

Impact of BK polyomavirus viremia on the outcomes of allogeneic hematopoietic stem cell transplantation.

Although it is known that BK polyomavirus (BKPyV) causes hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT), the clinical significance of BKPyV viremia has not been fully evaluated. We retrospectively analyzed the results of quantitative polymerase chain reaction (PCR) evaluations for detecting BKPyV in the whole blood samples of patients undergoing allogeneic HSCT during the period from January 2010 to June 2020 at a single institute, Tokyo Medical and Dental University. BKPyV was detected in the blood of 28 of the 107 evaluated patients, and the cumulative incidence of was 27.9% (95%CI: 20.2-37.9%). HC due to BKPyV developed in four of the 28 patients with BKPyV viremia (14.3%) and in two of the 79 patients without it (2.5%; P < 0.05). BKPyV viremia itself did not affect the patients' post-transplant estimated glomerular filtration rate (eGFR), but BKPyV viremia with a high viral load was significantly associated with decreased eGFR values (P < 0.05). BKPyV viremia was also associated with significantly lower progression-free survival at 3 years (35.1% [95%CI: 17.8-53.1%] vs. 60.4% [95%CI: 48.4-70.5], P < 0.05). Our findings demonstrated that BKPyV viremia was associated with onset of HC, an early decline of renal function, and poorer survival after allogeneic HSCT. Further studies are needed to test these results and elucidate the mechanisms of renal dysfunction associated with BKPyV viremia.

Lymphoproliferative disorder risk after methotrexate treatment for rheumatoid arthritis.

Methotrexate (MTX)-associated lymphoproliferative disorder (MTX-LPD) is a troublesome problem in patients receiving MTX for rheumatoid arthritis (RA). However, its incidence, prognosis, and risk factors remain unclear. In this retrospective study, we evaluated the actual incidence, prognostic impact, and risk factors of MTX-LPD. Of the 986 patients with RA treated with MTX, 90 patients experienced 95 new malignancies (NMs), with LPD as the most frequent in 26 patients. The cumulative LPD incidences were 1.3% and 4.7% at 5 and 10 years after MTX initiation, respectively. Among the 24 patients who discontinued MTX after developing LPD, 15 showed sustained regression, without difference in overall survival between patients with LPD and without NM. Inflammatory markers and absolute lymphocyte counts were not useful for early LPD development detection, but most of the patients with LPD had persistently elevated erythrocyte sedimentation ratios. Regarding concomitant drugs, tacrolimus increased the risk only if patients were not receiving biological disease-modifying antirheumatic drugs (bDMARDs). bDMARDs did not increase the risk for any of the drugs or the number of classes used. The number of LPD cases was lower in patients with IL-6A even after a long period after MTX, although with no statistically significant difference. Thus, approximately 1 in 20 patients with RA developed MTX-LPD over the 10 years of MTX treatment, but it did not affect the survival of patients with RA. Tacrolimus increased the risk of developing LPD for certain patients and should be used with caution.

[Acute promyelocytic leukemia with asymptomatic cerebral hemorrhage].

A 43-year-old man presenting with oral bleeding was diagnosed with acute promyelocytic leukemia (APL). Induction chemotherapy consisting of all-trans retinoic acid and idarubicin was initiated, and disseminated intravascular coagulation (DIC) was treated with fresh frozen plasma and recombinant thrombomodulin infusions. The patient was free from neurological symptoms throughout the clinical course. However, cerebral hemorrhagic lesions were detected incidentally on magnetic resonance imaging performed to screen for leukemic central nervous system invasion at 2 weeks after treatment initiation. Imaging findings suggested subacute or later-phase cerebral hemorrhage. Platelet transfusions and other supportive care was provided. Serial imaging evaluations confirmed reduction of the hemorrhagic lesions. Hematological remission was achieved after induction chemotherapy, and no symptoms due to cerebral hemorrhage developed during the subsequent consolidation therapy. As patients with APL characteristically experience hemorrhagic events due to bleeding tendency caused by DIC, physicians should be aware of the possibility of asymptomatic cerebral hemorrhage in these patients.

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.

[Malignant paraganglioma mimicking multiple myeloma].

We here present a 33-year-old woman who was referred to our hospital with a complaint of back pain and was found to have elevated IgG and hypercalcemia, as well as osteolytic lesions of pelvis and spines. 18F-FDG-PET/CT scan revealed numerous uptakes in the bones. An examination of the bone marrow revealed increased plasma cells (10.2%). Despite clinical similarities to multiple myeloma, any evidence of plasma cell clonal proliferation, including serum M-protein and light chain restriction, was not found. A reexamination of the bone marrow with a biopsy revealed the proliferation of abnormal cells with chromogranin A and synaptophysin expression but no expression of hematopoietic and epithelial cell markers. Based on these results together with extra-adrenal lesions, a diagnosis of malignant paraganglioma was made. Malignant paraganglioma is known to frequently cause bone metastasis and skeletal related events, whose clinical manifestations are similar to those of multiple myeloma. Since patients with osteolytic lesions, hypercalcemia, and hypergammaglobulinemia are likely to be referred to hematologists, malignant paraganglioma should be considered as a differential diagnosis.

[Perioperative management of laparoscopic cholecystectomy in a patient with paroxysmal nocturnal haemoglobinuria undergoing ravulizumab treatment].

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis, thrombosis, and bone marrow failure. Infection, pregnancy, and surgical operation have the potential to evoke severe episodes of hemolysis and thrombosis. Therefore, the use of an antibody agent against complement component 5 (C5), eculizumab, one day before the operation is recommended. Ravulizumab is a newly approved long-acting antibody agent against C5. Thus, little is known about perioperative management with ravulizumab. We experienced a 43-year-old female patient who safely underwent laparoscopic cholecystectomy under ravulizumab treatment for PNH. Ravulizumab was administered one day before the operation. Laparoscopic cholecystectomy for cholelithiasis was performed under intravenous anesthesia, intermittent air compression of the lower extremities, and low pneumoperitoneum pressure. Additionally, heparin was administered, and the patient left the sickbed early without significant postoperative complications. Like eculizumab, complement inhibition by ravulizumab is also considered effective in the perioperative management of patients with PNH. However, close cooperation with surgeons and anesthesiologists and careful management based on clinical symptoms and laboratory data such as LDH, CH50, and D-dimer are essential.

[Marginal zone lymphoma-like primary bone marrow lymphoma with long-term pancytopenia preceding diagnosis].

A 45-year-old man initially diagnosed with aplastic anemia had been receiving treatment for >4 years when he visited our hospital for a detailed examination. On admission, bone marrow (BM) aspiration showed erythroid dysplasia and chromosomal abnormalities, including trisomy 3 in 1/20 cells. After 3 months of observation, BM aspiration showed the involvement of 5% abnormal lymphocytes, and flow cytometry revealed a monoclonal B-cell phenotype. After a further 5 months of observation, his blood test showed a sudden elevation in white blood cell (WBC) count and the presence of villous lymphocytes. Fluorodeoxyglucose-positron emission tomography (FDG-PET) only revealed strong uptake by systemic BM, and BM aspiration showed the involvement of 76.4% abnormal lymphocytes, which were positive for CD19 and dim CD11c; negative for CD25, CD103, cyclin D1, and BRAF-V600E; and exhibited light chain restriction. The patient was diagnosed with marginal zone lymphoma-like primary bone marrow (BM) lymphoma. Treatment with R-CHOP and R-cladribine failed. He then underwent an allogeneic peripheral blood stem cell transplantation from a human leucocyte antigen (HLA)-identical sibling, and he has since remained in good health and without relapse for 9 years. Further clinical and biological analyses are necessary to establish an optimal treatment strategy for this disease.

[Impaired hematopoiesis due to copper deficiency in a hemodialysis patient supplemented with zinc].

This is a case of a 75-year-old man who was on maintenance hemodialysis for 10 years due to diabetic nephropathy and was prescribed polaprezinc due to a low serum zinc level (55 µg/dl) and dysgeusia. Three months after the polaprezinc treatment was initiated, the patient developed pancytopenia, which persisted even after the serum zinc level was normalized and medication was discontinued. He was referred to our institute so that the progression of pancytopenia could be assessed. A blood biochemical examination revealed a WBC count of 1,700/µl, Hb level of 8.9 g/dl, and Plt count of 9.5×104/µl. A bone marrow aspirate smear showed slight megaloblastic changes and ringed sideroblasts in addition to an elevated WT1 mRNA level (76 copies/µg RNA) in the peripheral blood. Although these findings mimicked those of myelodysplasia, low serum copper (<2 µg/dl) and ceruloplasmin levels (3 mg/dl) were suggestive of hematopoietic abnormalities due to zinc-induced copper deficiency. Treatment with cocoa, a compound generally known to be rich in copper, gradually improved the pancytopenia and dysplastic bone marrow histology. This case indicates that clinicians should consider the risk of zinc-induced copper deficiency and its complications when zinc supplementation is administered to patients with chronic kidney disease, particularly those undergoing hemodialysis.

[Follicular lymphoma accompanied by paraneoplastic pemphigus and bronchiolitis obliterans: a case report].

A 54-year-old female complained of oral erosion. A flaccid blister appeared on the trunk 2 months after the onset. The high titer of the anti-desmoglein 1 antibody in the absence of Nikolsky's sign led to the diagnosis of pemphigus vulgaris. The lymphadenopathy in the mesenteric and para-aortic regions indicated the possibility of paraneoplastic pemphigus. The steroid pulse therapy and therapeutic plasma exchange were ineffective. As CT-guided intraperitoneal lymph node biopsy revealed follicular lymphoma, R-CHOP therapy was performed. Although partial remission was attained accompanied by an improvement in the skin and mucosal findings after four courses of R-CHOP therapy, an occlusive ventilatory disturbance, possibly attributed to bronchiolitis obliterans, appeared 4 months after the treatment initiation. Although the treatment with tacrolimus was attempted, it was not feasible to be continued because of opportunistic infection, and the patient died 9 months after the onset of the skin lesion. Although specific anti-plakin antibodies were negative, this case was diagnosed as paraneoplastic pemphigus due to follicular lymphoma and complicated by obstructive bronchiolitis based on the clinical findings. The accumulation of similar cases is needed to establish effective treatment strategies.

Molecular mechanisms for enhancement of stromal cell-derived factor 1-induced chemotaxis by platelet endothelial cell adhesion molecule 1 (PECAM-1).

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration. Interestingly, several PECAM-1-deficient hematopoietic cells exhibit impaired chemotactic responses to stromal cell-derived factor 1 (SDF-1), a chemokine essential for B lymphopoiesis and bone marrow myelopoiesis. However, whether PECAM-1 is involved in SDF-1-regulated chemotaxis is unknown. We report here that SDF-1 induces tyrosine phosphorylation of PECAM-1 at its immunoreceptor tyrosine-based inhibition motifs in several hematopoietic cell lines via the Src family kinase Lyn, Bruton's tyrosine kinase, and JAK2 and that inhibition of these kinases reduced chemotaxis. Overexpression and knockdown of PECAM-1 enhanced and down-regulated, respectively, SDF-1-induced Gαi-dependent activation of the PI3K/Akt/mTORC1 pathway and small GTPase Rap1 in hematopoietic 32Dcl3 cells, and these changes in activation correlated with chemotaxis. Furthermore, pharmacological or genetic inhibition of the PI3K/Akt/mTORC1 pathway or Rap1, respectively, revealed that these pathways are independently activated and required for SDF-1-induced chemotaxis. When coexpressed in 293T cells, PECAM-1 physically associated with the SDF-1 receptor CXCR4. Moreover, PECAM-1 overexpression and knockdown reduced and enhanced SDF-1-induced endocytosis of CXCR4, respectively. Furthermore, when expressed in 32Dcl3 cells, an endocytosis-defective CXCR4 mutant, CXCR4-S324A/S325A, could activate the PI3K/Akt/mTORC1 pathway as well as Rap1 and induce chemotaxis in a manner similar to PECAM-1 overexpression. These findings suggest that PECAM-1 enhances SDF-1-induced chemotaxis by augmenting and prolonging activation of the PI3K/Akt/mTORC1 pathway and Rap1 and that PECAM-1, at least partly, exerts its activity by inhibiting SDF-1-induced internalization of CXCR4.

Characterization of three strains of Capnocytophaga canis isolated from patients with sepsis.

Capnocytophaga canimorsus and Capnocytophaga cynodegmi, both commensal bacteria in the oral cavities of dogs and cats, are zoonotic pathogens. In particular, C. canimorsus causes sepsis and fatal septic shock. Recently, a novel Capnocytophaga species, C. canis, was isolated from the oral cavities of healthy dogs. It is reportedly oxidase-negative and therefore considered avirulent in humans. In the present study, three strains of C. canis were isolated from Japanese patients with sepsis. All three strains, HP20001, HP33001 and HP40001, were oxidase-positive. Nucleotide sequence identities of the 16S rRNA gene of the three strains to the C. canimorsus type strain ATCC35979, C. cynodegmi type strain ATCC49044 and C. canis type strain LMG29146 were 96.9-97.0%, 96.9-97.0% and 99.7-99.8%, respectively. Multi-locus sequence analysis based on seven house-keeping genes, dnaJ, fumC, glyA, gyrB, murG, trpB and tuf, revealed that the oxidase-positive C. canis strains isolated in Japan and oxidase-negative strains of C. canis from canine oral cavities in Switzerland were clustered in different genetic subgroups. These results indicate that the virulence of C. canis strains in humans is associated with oxidase activity.

[A fatal case of septicemia by serotype A:3 Pasteurella multocida in human: review of 14 septic cases in Japan].

We report a case of systemic inflammatory response syndrome (SIRS) due to septicemia by Pasteurella multocida (P. multocida) who died three days after on admission. A few cases of septicemia by P. multocida were previously reported. In particular, capsular serotype analysis and somatic serotype analysis of P. multocida in human is very rare. Serotype A:3 P. multocida was identified in a diabetic patient with chronic obstructive pulmonary disease (COPD). There was high possibility that the isolate has been transmitted from an indoor companion dog since the patient had had close contact with the dog for one month. Here we discuss our case and 13 human cases of septicemia by P. multocida reported in Japan. Four cases from dogs and eleven cases from cats were included in these cases. The mortality rate of septicemia by P. multocida was 21.4% (3/14 cases). Two out of three cases died were diabetic.

Establishment and characterization of a new activated B-cell-like DLBCL cell line, TMD12.

We report the establishment of a novel activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) cell line, designated as TMD12, from a patient with highly refractory DLBCL. ABC-DLBCL is a subtype with a relatively unfavorable prognosis that was originally categorized using gene expression profiling according to its cell of origin. TMD12 cells were isolated from the pleural effusion of the patient at relapse and passaged continuously in vitro for >4 years. The cells displayed cluster of differentiation (CD)19, CD20, CD22, CD38, human leukocyte antigen-DR isotype, and κ positivity and CD5, CD10, CD23, and λ negativity, as detected using flow cytometric analysis. The chromosomal karyotypic analysis, including the spectral karyotyping method, confirmed t(1;19)(q21:q13.1), del(6q23), gain of chromosome 18, and other abnormalities. Mutation analyses, including whole-exome sequencing, revealed that TMD12 cells harbored mutations in MYD88 and CD79B, indicating an ABC subtype. TMD12 cells exhibited chronic active B-cell receptor signaling and constitutive activation of the nuclear factor κB pathway, which is typically associated with sensitivity to a specific Bruton tyrosine kinase inhibitor, ibrutinib. Intriguingly, TMD12 cells displayed moderate resistance to ibrutinib and lacked activation of Janus kinase/signal transducers and activators of transcription 3 signaling, another hallmark of this DLBCL subtype. Treatment with an inhibitor against tumor progression locus 2 (TPL2), a multifunctional intracellular kinase that is activated particularly downstream of Toll-like receptors or MYD88 and IκB kinase α/ß (IKKα/ß), suppressed the proliferation of TMD12 cells, implying the possible involvement of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL. Because only a limited number of ABC-DLBCL cell lines are currently available, TMD12 cells might provide a useful tool in the search for novel druggable targets for this intractable lymphoma.

A novel chimeric antigen receptor (CAR) system using an exogenous protease, in which activation of T cells is controlled by expression patterns of cell­surface proteins on target cells.

Anti­CD19 chimeric antigen receptor (CAR)­T cell therapy against refractory B­cell malignancies shows excellent therapeutic effects. However, there are some obstacles to be overcome in this treatment. Since current CAR­T cells target a single cell­surface protein on tumor cells, the CAR­T cells also attack normal cells expressing the protein. This is one of the major adverse effects of this therapy. To improve target­cell­specificity of this therapy, we established a novel CAR system, in which T­cell activation was controlled by expression patterns of proteins on target cells. Our novel CAR­T cells had two distinct CARs consisting of a 'Signal­CAR', recognizing a protein on tumor cells, and a 'Scissors­CAR', recognizing another protein on normal cells. The signal­CAR had a peptide sequence which was cleaved by the Scissors­CAR, and functional domains for cellular activation. The Scissors­CAR had a protease domain that cleaved its recognition peptide sequence in the Signal­CAR. When tumor cells expressed only the protein recognized by the Signal­CAR, the tumor cells were attacked. By contrast, normal cells expressing both the proteins induced inactivation of the Signal­CAR through cleavage of the recognition site when getting in contact with the CAR­T cells. To establish this system, we invented a Scissors­CAR that was dominantly localized on cell membranes and was activated only when the CAR­T cells were in contact with the normal cells. Using a T­cell line, Jurkat, and two proteins, CD19 and HER2, as target proteins, we showed that the anti­CD19­Signal­CAR was cleaved by the anti­HER2­Scissors­CAR when the CAR­T cells were co­cultivated with cells expressing both the proteins, CD19 and HER2. Furthermore, we demonstrated that primary CAR­T cells expressing both the CARs showed attenuated cytotoxicity againsT cells with both the target proteins. Our novel system would improve safety of the CAR­T cell therapy, leading to expansion of treatable diseases by this immunotherapy.

Severe Motor Weakness Due to Disturbance in Peripheral Nerves Following Tisagenlecleucel Treatment.

BACKGROUND: Neurotoxicity is one of the dangerous complications of chimeric antigen receptor (CAR) T-cell therapy, while its pathophysiology remains to be fully understood. Motor weakness not associated with central nervous system (CNS) toxicity has rarely been reported after CAR T-cell therapy. CASE REPORT: A 42-year-old female with a refractory diffuse large B-cell lymphoma received tisagenlecleucel (tisa-cel) and developed cytokine release syndrome (CRS) on day 3. She was treated with tocilizumab and methylprednisolone, which resolved CRS promptly. On day 7, motor weakness in lower extremities appeared, and she gradually became unable to walk without showing any other symptoms attributed to CNS disturbances. Whereas dexamethasone and tocilizumab were ineffective, neuropathy improved after high dose chemotherapy followed by autologous stem cell transplantation. Nerve conduction study (NCS) in lower extremities showed a decline in compound muscle action potential amplitude along with worsening of motor weakness, which was restored after improvement of symptoms. Based on symptoms and NCS, her motor weakness was thought to be due to disturbance in peripheral nerves. CONCLUSION: This study reports a patient who developed severe motor weakness due to disturbance in peripheral nerves after tisa-cel therapy. Neurotoxicity of non-CNS origin should also be noted in CAR T-cell therapy.

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.

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 induces apoptosis in FLT3-ITD-positive AML cells cooperatively by inhibiting the mutant kinase through aggresomal translocation and inducing oxidative stress.

FLT3-ITD and FLT3-TKD are the most frequent mutations in acute myeloid leukemia (AML) with the former associated with a poor prognosis. Here we show that inhibition of the deubiquitinase USP9X by its inhibitor WP1130 or EOAI3402143 (G9) induces apoptosis preferentially in cells transformed by these mutant kinases, including FLT3-ITD-positive AML cell line MV4-11 and primary AML cells. Mechanistically, WP1130 induced aggresomal translocation of the mutant kinases, particularly FLT3-ITD in its activated and autophosphorylated conformation, to block the downstream signaling events, which was aggravated by knock down of USP9X. Moreover, USP9X physically associated with FLT3-ITD to inhibit its K63-linked polyubiquitination, while FLT3-ITD induced tyrosine phosphorylation and degradation of USP9X through the ubiquitin/proteasome pathway. WP1130 or G9 also induced oxidative stress to stimulate stress-related MAP kinase pathways and DNA damage responses to activate in cooperation with inhibition of FLT3-ITD signaling the intrinsic mitochondria-mediated apoptotic pathway, which was synergistically enhanced by BH3 mimetics and prevented by overexpression of Bcl-xL or Mcl-1. Thus, USP9X represents a promising target for novel therapies against therapy-resistant FLT3-ITD-positive AML.

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.