Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment.

Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.

IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

CHIP-associated mutant ASXL1 in blood cells promotes solid tumor progression.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid-tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock-in mice that express CHIP-associated mutant Asxl1 (Asxl1-MT), we showed that expression of Asxl1-MT in T cells, but not in myeloid cells, promoted solid-tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1-MT-expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV-PyMT. Intratumor analysis of the mammary tumors revealed the reduced T-cell infiltration at tumor sites and programmed death receptor-1 (PD-1) upregulation in CD8+ T cells in MMTV-PyMT/Asxl1-MT mice. In addition, we found that Asxl1-MT induced T-cell dysregulation, including aberrant intrathymic T-cell development, decreased CD4/CD8 ratio, and naïve-memory imbalance in peripheral T cells. These results indicate that Asxl1-MT perturbs T-cell development and function, which contributes to creating a protumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1-mutated blood cells exacerbate solid-tumor progression in ASXL1-CHIP carriers.

CD38 expression is an important prognostic marker in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases with variable outcomes. Although several prognostic markers have been developed, specific biomarkers for stratifying treatment strategies have not been fully investigated. This study aimed to analyze the clinical impact of the expression of cluster of differentiation (CD) 38, which is associated with cellular proliferation and disease progression, in patients with de-novo DLBCL. Using flow cytometry analysis, 137 cases with DLBCL were investigated for surface expression of CD38. Based on the cut-off value by the survival classification and regression tree analysis, the patients were categorized into a CD38HIGH group (n = 37) and CD38LOW group (n = 100). The 4-years progression-free survival (PFS) was 31.6% in the CD38HIGH group and 60.7% in the CD38LOW group (p < 0.001). Multivariate analysis showed the CD38HIGH group to be associated with significantly worse PFS (adjusted hazard ratio [aHR], 2.15, 95% CI: 1.26-3.68, p = 0.005) and poor overall survival (OS) (aHR, 2.54, 95% CI: 1.25-5.19, p = 0.010) than the CD38LOW group. In conclusion, we demonstrated that high CD38 expression is an independent adverse prognostic factor associated with poor clinical outcomes compared to low CD38 expression. CD38 expression in DLBCL cells might be useful for predicting outcomes and designing risk-adapted therapies for patients with de-novo DLBCL.

The impact of early diagnosis on the prognosis of extranodal NK/T-cell lymphoma with massive lung involvement: a case report.

BACKGROUND: Pulmonary non-Hodgkin lymphoma (NHL) is rare. The most frequent subtype of pulmonary NHL is low-grade B-cell lymphoma, such as lymphoma of mucosa-associated lymphoma tissue. Extranodal natural killer cell/T-cell lymphoma, nasal type (ENKL) is characterized by predominant extranodal involvement and association with Epstein-Barr virus (EBV). ENKL with massive lung involvement has been infrequently reported, and its prognosis is extremely poor. CASE PRESENTATION: A 20-year-old Japanese man presented with intermittent fever lasting for 2 months. Radiological imaging demonstrated multiple nodules of uneven shape and size in both lungs. Video-assisted thoracic surgical lung biopsy showed abnormal lymphocyte infiltration, which was positive for CD3, CD56, and perforin. In situ hybridization for EBV-encoded RNA was positive. From these findings, he was diagnosed with ENKL with lung involvement. The patient was successfully treated with intensive combinational chemotherapy followed by allogeneic cord blood transplantation. He has been alive with continuous complete remission for 1 year after diagnosis. CONCLUSIONS: Although ENKL involving the lung has been reported to have dismal outcomes, our patient showed long-term survival after intensive chemotherapy and up-front allogeneic hematopoietic transplantation. The present case highlights the importance of early diagnosis as well as allogeneic transplantation.

ASXL1 and SETBP1 mutations promote leukaemogenesis by repressing TGFß pathway genes through histone deacetylation.

Mutations in ASXL1 and SETBP1 genes have been frequently detected and often coexist in myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We previously showed that coexpression of mutant ASXL1 and SETBP1 in hematopoietic progenitor cells induced downregulation of TGFß pathway genes and promoted the development of MDS/AML in a mouse model of bone marrow transplantation. However, whether the repression of TGFß pathway in fact contributes to leukaemogenesis remains unclear. Moreover, mechanisms for the repression of TGFß pathway genes in ASXL1/SETBP1-mutated MDS/AML cells have not been fully understood. In this study, we showed that expression of a constitutively active TGFß type I receptor (ALK5-TD) inhibited leukaemic proliferation of MDS/AML cells expressing mutant ASXL1/SETBP1. We also found aberrantly reduced acetylation of several lysine residues on histone H3 and H4 around the promoter regions of multiple TGFß pathway genes. The histone deacetylase (HDAC) inhibitor vorinostat reversed histone acetylation at these promoter regions, and induced transcriptional derepression of the TGFß pathway genes. Furthermore, vorinostat showed robust growth-inhibitory effect in cells expressing mutant ASXL1, whereas it showed only a marginal effect in normal bone marrow cells. These data indicate that HDAC inhibitors will be promising therapeutic drugs for MDS and AML with ASXL1 and SETBP1 mutations.

[Mitoxantrone, etoposide, and cytarabine combination therapy for acute myeloid leukemia patients failing to achieve a complete remission after induction chemotherapy: a single-center experience].

Optimal salvage chemotherapy has not been established for patients with acute myeloid leukemia (AML) who fail to attain complete remission (CR) after one course of induction chemotherapy. This retrospective study aimed to assess the efficacy and safety of an MEC (mitoxantrone, 6 mg/m2, 1-3 days; etoposide, 80 mg/m2, 1-6 days; cytarabine, 1 g/m2, 1-6 days) regimen in patients with AML who failed to attain CR after one course of induction chemotherapy. Twenty-four patients were included in this study (median age, 58 years; range, 28-79 years). After one course of MEC, 11 patients (45.8%) attained CR. Febrile neutropenia was observed in all patients, and acute infection was observed in 7 patients (29.2%). However, no therapy-related death occurred. All patients eligible for transplantation and who attained CR after MEC salvage chemotherapy underwent allogeneic hematopoietic stem cell transplantation. The MEC regimen exhibited a good response rate with tolerable adverse events. Therefore, the MEC regimen can be safely used as a salvage treatment for patients with AML who failed to attain CR after one course of induction chemotherapy.

Adult-onset primary cyclic autoimmune neutropenia: a case report.

BACKGROUND: A few cases of primary autoimmune neutropenia (AIN) have been reported in adults, but cyclic primary AIN, which is characterized by the periodic oscillation of neutrophils, is uncommon in adults. STUDY DESIGN AND METHODS: Herein, we report a 70-year-old man referred to our hospital with severe neutropenia and thrombocytopenia. He had experienced intermittent episodes of low-extremity purpura for the past 3 months, with cellulitis on the skin of the scalp 1 month previously. RESULTS: The patient presented with severely low neutrophil and platelet (PLT) counts. Myeloid progenitors and megakaryocytes were increased in the marrow, but mature neutrophils were remarkably decreased. Anti-neutrophil antibodies to specific epitopes were detected at neutropenia. Based on these findings, AIN accompanied by autoimmune thrombocytopenia was diagnosed. The patient experienced synchronous fluctuations of neutrophil and PLT counts three times. Despite no treatment, the neutrophil count fluctuated within the range of 0.06 × 109 to 1.65 × 109 /L, and the PLT count fluctuated from 0.7 × 1010 to 20.5 × 1010 /L. We identified an inverse relationship between neutrophil count and anti-neutrophil antibody titers, establishing the conclusive diagnosis of cyclic AIN. After prednisolone treatment, the neutrophil and PLT counts normalized, and the patient has maintained long-term remission. CONCLUSION: We report a rare case of cyclic AIN diagnosed from the inverse association between periodic oscillation of anti-neutrophil antibody titers and neutrophil counts. This clinical course suggests that in AIN patients, laboratory data and recurrent signs of infection should be monitored regularly, including shortly after neutrophil recovery.

Expression of multiple leukemic stem cell markers is associated with poor prognosis in de novo acute myeloid leukemia.

Leukemic stem cells (LSCs) play a crucial role in chemotherapy resistance in acute myeloid leukemia (AML). Although the association between the expression of individual LSC markers and poor prognosis has been reported, few studies have evaluated the prognostic effect of multiple LSC markers in patients with AML. Herein, we examined three LSC markers (CD25, CD96, and CD123) and the combined effect of their expression on clinical outcome. We retrospectively analyzed 80 adult patients with de novo AML who received intensive chemotherapy. Multiple LSC marker expression was significantly associated with shorter three-year overall survival (OS), compared with single or no LSC marker expression (18.2 vs. 65.0%, p < .001). Multivariate analysis showed that the expression of multiple LSC markers remained significant in terms of three-year OS (hazard ratio: 3.80, p = .001). Therefore, the combined evaluation of several LSC markers can predict the clinical outcome in patients with AML.

Successful Treatment of Herpes Simplex Virus (HSV)-1-associated Hemophagocytic Lymphohistiocytosis (HLH) with Acyclovir: A Case Report and Literature Review.

Hemophagocytic lymphohistiocytosis (HLH) associated with herpes simplex virus (HSV)-1 infection (HSV-1-HLH) is uncommon and is potentially fatal without appropriate treatment. We herein report the case of an adult patient with HSV-1-HLH who was successfully treated with acyclovir. A 69-year-old man developed fever, pancytopenia and liver enzyme elevation after the resolution of pneumonia. These findings and the presence of hemophagocytosis in the patient's bone marrow were consistent with a diagnosis of HLH. The patient was diagnosed with HSV-1-HLH based on the results of a polymerase chain reaction (PCR) for HSV-1. The early administration of acyclovir improved his clinical symptoms and laboratory results within two weeks. In the present case, the rapid and precise diagnosis facilitated the successful treatment of HSV-1-HLH.

Peripheral Blood Lymphocyte-to-Monocyte Ratio at Relapse Predicts Outcome for Patients With Relapsed or Refractory Diffuse Large B-cell Lymphoma in the Rituximab Era.

BACKGROUND: Patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) have a poor prognosis, even in the rituximab era. Several studies have reported the clinical importance of the peripheral blood lymphocyte-to-monocyte ratio (LMR) in various malignancies, including lymphoma. However, the prognostic value of the LMR in relapsed/refractory DLBCL has not been well evaluated. The purpose of the present study was to investigate whether the LMR at relapse can predict clinical outcomes for relapsed/refractory DLBCL patients treated with rituximab. PATIENTS AND METHODS: We analyzed data on 74 patients with relapsed/refractory DLBCL, who were initially treated with R-CHOP (rituximab and cyclophosphamide, doxorubicin, vincristine, and prednisone) or an R-CHOP-like regimen. RESULTS: There was a significant association between a low LMR (≤ 2.6) and shorter overall survival (OS; P < .001) and progression-free survival (PFS; P < .001) compared with the high LMR group (> 2.6). Multivariate analysis showed that LMR was an independent prognostic factor for OS (P < .001) and PFS (P < .001), as was the international prognostic index (IPI) at relapse for OS. In addition, the LMR had an incremental value for OS and PFS compared with the IPI at relapse. CONCLUSION: The LMR predicts OS and PFS outcomes in relapsed/refractory DLBCL patients treated with rituximab, and might facilitate better stratification among patients in low- and intermediate-risk IPI groups.

[Durable remission attained by long-term brentuximab vedotin administration in a relapsed post-allogeneic bone marrow transplant Hodgkin lymphoma patient].

The prognosis for relapsed Hodgkin lymphoma after allogeneic hematopoietic cell transplantation (HSCT) is poor, partly because of limited treatment options. Here we present a case of a Hodgkin lymphoma patient who relapsed after allogeneic HSCT but remains in complete remission (CR) at 38 months from the start of extended brentuximab vedotin (BV) dosing. A 33-year-old man with refractory and relapsed nodular sclerosis classical Hodgkin lymphoma who underwent previous treatments, including adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) ; seven combination regimens; and autologous HSCT, prior to allogeneic HSCT achieved CR after three cycles of BV. BV was continued for 26 cycles and then discontinued because of a neurogenic bladder. The other adverse effects were mild paresthesia in the fingers, mild dysgeusia, and fatigue. The patient still remains in CR at 38 months from the start of BV. Thus, extended BV dosing may be a treatment option for relapsed and refractory Hodgkin lymphoma after allogeneic HSCT.