Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs.

Despite evidence of chronic inflammation in myelodysplastic syndrome (MDS) and cell-intrinsic dysregulation of Toll-like receptor (TLR) signaling in MDS hematopoietic stem and progenitor cells (HSPCs), the mechanisms responsible for the competitive advantage of MDS HSPCs in an inflammatory milieu over normal HSPCs remain poorly defined. Here, we found that chronic inflammation was a determinant for the competitive advantage of MDS HSPCs and for disease progression. The cell-intrinsic response of MDS HSPCs, which involves signaling through the noncanonical NF-κB pathway, protected these cells from chronic inflammation as compared to normal HSPCs. In response to inflammation, MDS HSPCs switched from canonical to noncanonical NF-κB signaling, a process that was dependent on TLR-TRAF6-mediated activation of A20. The competitive advantage of TLR-TRAF6-primed HSPCs could be restored by deletion of A20 or inhibition of the noncanonical NF-κB pathway. These findings uncover the mechanistic basis for the clonal dominance of MDS HSPCs and indicate that interfering with noncanonical NF-κB signaling could prevent MDS progression.

The pathogenetic significance of exhausted T cells in a mouse model of mature B cell neoplasms.

Dysfunctional anti-tumor immunity has been implicated in the pathogenesis of mature B cell neoplasms, such as multiple myeloma and B cell lymphoma; however, the impact of exhausted T cells on disease development remains unclear. Therefore, the present study investigated the features and pathogenetic significance of exhausted T cells using a mouse model of de novo mature B cell neoplasms, which is likely to show immune escape similar to human patients. The results revealed a significant increase in PD-1+ Tim-3- and PD-1+ Tim-3+ T cells in sick mice. Furthermore, PD-1+ Tim-3+ T cells exhibited direct cytotoxicity with a short lifespan, showing transcriptional similarities to terminally exhausted T cells. On the other hand, PD-1+ Tim-3- T cells not only exhibited immunological responsiveness but also retained stem-like transcriptional features, suggesting that they play a role in the long-term maintenance of anti-tumor immunity. In PD-1+ Tim-3- and PD-1+ Tim-3+ T cells, the transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in vivo. These transcription factors were down-regulated by IMiDs in our in vitro T cell exhaustion analyses. The prevention of excessive T cell exhaustion may maintain effective anti-tumor immunity to cure mature B cell neoplasms.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

[Role of innate immune signaling and inflammation in the pathogenesis of myeloid malignancies].

Myeloid malignancies are composed of multiple clonal hematopoietic disorders, including myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia. Inflammation is already known to play an important role in the pathogenesis of an extensive variety of malignancies, and its significance in myeloid malignancies is becoming more widely recognized. Specifically, cell-intrinsic and -extrinsic activation of the innate immune signaling pathway, as well as elevation of proinflammatory cytokines via innate immune signaling downstream signaling, have been demonstrated. Furthermore, the inflammatory microenvironment refers to the bone marrow environment rich in inflammatory signaling molecules that surround hematopoietic malignant cells, and its role in the pathogenesis of myeloid malignancies has been extensively studied in recent years. Herein, we present the latest findings and discuss how innate immune signaling activation and the inflammatory bone marrow microenvironment contribute to the pathogenesis of myeloid malignancies.

Genetic subtype classification using a simplified algorithm and mutational characteristics of diffuse large B-cell lymphoma in a Japanese cohort.

Recent large-scale genetic studies have proposed a new genetic classification of diffuse large B-cell lymphoma (DLBCL), which is clinically and biologically heterogeneous. However, the classification methods were complicated to be introduced into clinical practice. Here we retrospectively evaluated the mutational status and copy number changes of 144 genes in 177 Japanese patients with DLBCL, using targeted DNA sequencing. We developed a simplified algorithm for classifying four genetic subtypes-MYD88, NOTCH2, BCL2, and SGK1-by assessing alterations in 18 representative genes and BCL2 and BCL6 rearrangement status, integrating the significant genes from previous studies. In our cohort and another validation cohort from published data, the classification results in our algorithm showed close agreement with the other established algorithm. A differential prognosis among the four groups was observed. The NOTCH2 group showed a particularly poorer outcome than similar groups in previous reports. Furthermore, our study revealed unreported genetic features in the DLBCL subtypes that are mainly reported in Japanese patients, such as CD5-positive DLBCL and methotrexate-associated lymphoproliferative disorders. These results indicate the utility of our simplified method for DLBCL genetic subtype classification, which can facilitate the optimisation of treatment strategies. In addition, our study highlights the genetic features of Japanese patients with DLBCL.

Long-term efficacy of partial splenic embolization for the treatment of steroid-resistant chronic immune thrombocytopenia.

Thrombopoietin-receptor agonists have been recently introduced for a second-line treatment of immune thrombocytopenia (ITP). Splenectomy has tended to be avoided because of its complications, but the response rate of splenectomy is 60-80% and it has still been considered for steroid-refractory ITP. We performed partial splenic embolization (PSE) as an alternative to splenectomy. Between 1988 and 2013, 91 patients with steroid-resistant ITP underwent PSE at our hospital, and we retrospectively analyzed the efficacy and long-term outcomes of PSE. The complete response rate (CR, platelets > 100 × 109/L) was 51% (n = 46), and the overall response rate (CR plus response (R), > 30 × 109/L) was 84% (n = 76). One year after PSE, 70% of patients remained CR and R. The group with peak platelet count after PSE ≥ 300 × 109/L (n = 29) exhibited a significantly higher platelet count than the group with platelet count < 300 × 109/L (n = 40) at any time point after PSE. The failure-free survival (FFS) rates at 1, 5, and 10 years were 78, 56, and 52%, respectively. Second PSE was performed in 20 patients who relapsed (n = 14) or had no response to the initial PSE (n = 6), and the overall response was achieved in 63% patients. There were no PSE-related deaths. These results indicate that PSE is a safe and effective alternative therapy to splenectomy for patients with steroid-resistant ITP as it generates long-term, durable responses.

Safety and Efficacy of Granulocyte Colony-Stimulating Factor Monotherapy for Peripheral Blood Stem Cell Collection in POEMS Syndrome.

Although autologous stem cell transplantation can achieve excellent responses in patients with POEMS syndrome, the optimal regimen for peripheral blood stem cell (PBSC) collection is still controversial. We retrospectively investigated the safety and efficacy of 41 PBSC collecting procedures in 37 patients with POEMS syndrome. PBSC mobilization was performed using cyclophosphamide + granulocyte colony-stimulating factor (G-CSF) (CG, n = 14) or G-CSF alone (G, n = 27). Twelve (85.7%) patients in the CG group and all (100%) patients in the G group received induction chemotherapy before PBSC collection. The proportions of good mobilizers (≥2.0 × 106 CD34+ cells/kg) were comparable between the 2 groups (CG versus G: 78.6% versus 70.4%, P = .71). Two (14.3%) patients in the CG group developed severe capillary leak symptoms during the PBSC mobilization period, whereas no patient in the G group experienced severe adverse events. Appropriate induction therapies followed by the G-CSF monotherapy compose an optimal strategy for PBSC collection.

Ezh2 loss in hematopoietic stem cells predisposes mice to develop heterogeneous malignancies in an Ezh1-dependent manner.

Recent genome sequencing revealed inactivating mutations in EZH2, which encodes an enzymatic component of polycomb-repressive complex 2 (PRC2), in patients with myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), and MDS/MPN overlap disorders. We herein demonstrated that the hematopoietic-specific deletion of Ezh2 in mice induced heterogeneous hematopoietic malignancies. Myelodysplasia was detected in mice following the deletion of Ezh2, and resulted in the development of MDS and MDS/MPN. Thrombocytosis was induced by Ezh2 loss and sustained in some mice with myelodysplasia. Although less frequent, Ezh2 loss also induced T-cell acute lymphoblastic leukemia and the clonal expansion of B-1a B cells. Gene expression profiling showed that PRC2 target genes were derepressed upon the deletion of Ezh2 in hematopoietic stem and progenitor cells, but were largely repressed during the development of MDS and MDS/MPN. Chromatin immunoprecipitation-sequence analysis of trimethylation of histone H3 at lysine 27 (H3K27me3) revealed a compensatory function of Ezh1, another enzymatic component of PRC2, in this process. The deletion of Ezh1 alone did not cause dysplasia or any hematologic malignancies in mice, but abolished the repopulating capacity of hematopoietic stem cells when combined with Ezh2 loss. These results clearly demonstrated an essential role of Ezh1 in the pathogenesis of hematopoietic malignancies induced by Ezh2 insufficiency, and highlighted the differential functions of Ezh1 and Ezh2 in hematopoiesis.

Acutely deteriorated extravascular volume overload during peripheral blood stem cell mobilization in POEMS syndrome: A case series with cytokine analysis.

We describe two cases of polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes (POEMS) syndrome patients with deteriorated extravascular volume overload without increased levels of vascular endothelial growth factor after the administration of cyclophosphamide + granulocyte colony-stimulating factor for stem cell mobilization. We then measured the serum levels of 27 cytokines from these cases using a multiplex suspension array system. The analysis revealed the changes of cytokine profiles before cyclophosphamide + granulocyte colony-stimulating factor and after the development of capillary leak symptoms in both cases. This may improve our current level of understanding of the pathogenesis of POEMS syndrome not driven by vascular endothelial growth factor.

Severe stomatitis and ileocecal perforation developed after all-trans retinoic acid monotherapy in an HLA-B51-positive patient with acute promyelocytic leukemia.

A 34-year-old man who had been referred to our hospital was diagnosed with acute promyelocytic leukemia (APL). All-trans retinoic acid (ATRA), oral administration, was initiated. On day 25, he developed fever and respiratory distress with bilateral pulmonary infiltrates, suggesting differentiation syndrome (DS) caused by ATRA. These symptoms showed amelioration after discontinuing ATRA and initiating methylprednisolone. ATRA was re-started on day 29 at half the original dose because of residual APL blasts. The patient subsequently developed fever, severe stomatitis, and oropharyngeal ulcers, which persisted even after discontinuing ATRA. On day 48, he suddenly developed severe abdominal pain with free air, observable on an abdominal X-ray, and underwent emergency ileocecal resection. Pathological examination of the resected ileocecal intestines revealed multiple ulcers and perforations. No leukemic cell infiltration was observed. In this case, only ATRA was administered for APL treatment. These findings suggest that ileocecal ulcerations and perforations, as well as oropharyngeal ulcers, might have been caused by DS or ATRA. Furthermore, DNA typing of the HLA-B locus revealed that the patient had HLA-B51 associated with Behçet's disease. Therefore, hypercytokinemia with DS might have induced Behçet's disease-like symptoms, including stomatitis and ileocecal perforation, complications that are particularly observed in patients with HLA-B51.

[Mutations of epigenetic regulator genes and myeloid malignancies].

Recent genome studies have identified recurrent somatic mutations in various myeloid malignancies, including acute myeloid leukemia, myelodysplastic syndrome and myeloproliferative neoplasm. These mutations frequently occur in epigenetic regulator genes, and functions of the proteins encoded by these genes in hematopoietic cells have been extensively analyzed, as reported recently. It is noteworthy that several epigenetic regulator genes, such as DNMT3A, TET2 and ASXL1, have also been identified in pre-leukemic stem cells. As targeting pre-leukemic stem cells would be a promising therapeutic approach, further investigations of epigenetic abnormalities in hematopoietic cells are anticipated to lead to the development of novel epigenetic therapies. In this review, we discuss recent genetic and functional data regarding epigenetic regulator genes and the future landscape of this new research field.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression.

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

Detection of clonal plasma cells in POEMS syndrome using multiparameter flow cytometry.

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein [M-protein], and skin changes) is a rare systemic disorder characterized by various symptoms caused by underlying plasma cell (PC) dyscrasia. Detection of monoclonal PCs is mandatory for the diagnosis of POEMS syndrome; however, the usefulness of EuroFlow-based next-generation flow cytometry (EuroFlow-NGF) in POEMS syndrome for detecting monoclonal PCs in bone marrow (BM) and the gating strategy suitable for flow cytometry study of POEMS syndrome remain unknown. We employed EuroFlow-NGF-based single-tube eight-color multiparameter flow cytometry (MM-flow) and established a new gating strategy (POEMS-flow) to detect the monoclonal PCs in POEMS syndrome, gating CD38 broadly from dim to bright and CD45 narrowly from negative to dim compared to MM-flow. MM-flow detected monoclonal PCs in 9/25 (36.0%) cases, including 2/2 immunofixation electrophoresis (IFE)-negative cases (100%). However, POEMS-flow detected monoclonal PCs in 18/25 cases (72.0%), including 2/2 IFE-negative cases (100%). POEMS-flow detected monoclonal PCs with immunophenotypes of CD19- in 17/18 (94.4%). In six cases where post-treatment samples were available, the size of the clones was significantly reduced after the treatment (P = 0.031). POEMS-flow can enhance the identification rate of monoclonal PCs in POEMS syndrome and become a valuable tool for the diagnosis of POEMS syndrome.

Unraveling unique features of plasma cell clones in POEMS syndrome with single-cell analysis.

POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.

TRAF6 functions as a tumor suppressor in myeloid malignancies by directly targeting MYC oncogenic activity.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.

Blocking UBE2N abrogates oncogenic immune signaling in acute myeloid leukemia.

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

Pharmacokinetically guided, once-daily intravenous busulfan in combination with fludarabine for elderly AML/MDS patients as a conditioning regimen for allogeneic stem cell transplantation.

The efficacy of pharmacokinetically (PK) guided, once-daily administration of busulfan (BU) was evaluated in elderly patients with acute myeloid leukemia/myelodysplastic syndrome (AML/MDS). Twenty-one patients (median age 61) received 30 mg/m2 fludarabine for 6 days and BU for 4 days, starting from 3.2 mg/m2 and subsequently adjusted to the target area under the curve (AUC) of 6000 µmol-min/L. The median AUC of day 1 (AUC1), AUC4, and their average were 4871.3, 6021.0, and 5368.1 µmol-min/L, respectively. Veno-occlusive disease/sinusoidal obstructive syndrome (VOD/SOS) occurred in five patients (24%) but all recovered well. Four patients (20%) had non-infectious pulmonary complications (NIPCs). Patients with high AUC1 had frequent gastrointestinal adverse events, but similar incidence of VOD/SOS and NIPCs. Two-year overall survival (OS), non-relapse mortality (NRM), and relapse rates were 44.4%, 28.6%, and 29.1%, respectively. Patients with high AUC1 had significantly high NRM (57.1% vs. 14.3%, P = 0.04) and inferior OS (14.3% vs. 60.1%, P = 0.002), while patients with high AUC4 had a significantly low relapse rate (8.3% vs. 55.6%, P = 0.02). In conclusion, once-daily BU and a PK-guided dose intensification were beneficial for reducing relapse in elderly patients with AML/MDS. However, caution should be exercised as rapid BU dose elevation may contribute to NRM.

Successful allogeneic bone marrow transplantation after massive gastrointestinal bleeding in a patient with myelodysplastic syndrome associated with intestinal Behçet-like disease.

A 45-year-old woman was diagnosed with myelodysplastic syndrome (MDS) with trisomy 8 and Behçet-like disease (BLD) with multiple colorectal ulcers. Nonspecific inflammatory cells were infiltrated in the intestinal mucosa, whereas fluorescence in situ hybridization (FISH) analysis revealed only sporadic trisomy 8-positive cells. She presented massive lower gastrointestinal bleeding early after bone marrow transplantation but achieved long-term remission of both MDS and BLD. This is the first report of massive gastrointestinal bleeding after transplantation for MDS with BLD. Based on FISH analysis, dysregulation of systemic inflammation may be involved in BLD rather than direct invasion by trisomy 8-positive MDS clones.

The combination of the tubulin binding small molecule PTC596 and proteasome inhibitors suppresses the growth of myeloma cells.

The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.