[FLT3-ITD mutation-positive acute myeloid leukemia undergoing clonal transition with PTPN11 mutation at relapse].

A 28-year-old man was diagnosed with acute myelomonocytic leukemia. He achieved complete remission (CR) after two cycles of induction therapy. However, after consolidation therapy, bone marrow aspiration performed to prepare for allogeneic hematopoietic stem cell transplantation revealed disease relapse. Companion diagnostics confirmed the presence of the FLT3-ITD mutation. The patient received gilteritinib monotherapy and achieved CR. Subsequently, he underwent unrelated allogeneic bone marrow transplantation. One year after transplantation, the patient relapsed, and gilteritinib was resumed. However, the leukemia progressed, and panel sequencing using a next-generation sequencer showed that the FLT3-ITD mutation disappeared. A mutation in PTPN11, which regulates the RAS/MAPK signaling pathway, was also detected. Gilteritinib was discontinued, and the patient achieved CR with salvage chemotherapy. He underwent related haploidentical peripheral blood stem cell transplantation but died of relapse. This was a case in which genetic analysis revealed clonal transition and acquisition of resistance to treatment.

Mitochondrial dynamics as a pathobiological mediator of clonal myeloid disorders.

Myeloid malignancies, including myelodysplastic syndromes and acute myeloid leukemia, are a group of clonal hematopoietic stem cell (HSC) diseases. The incidence increases with global population aging. Genome sequencing uncovered mutational profiles in patients with myeloid malignancies and healthy elderly individuals. However, the molecular and cellular basis of disease development remains unclear. Accumulating evidence shows mitochondrial involvement in the pathogenesis of myeloid malignancies, aging-related HSC phenotypes, and clonal hematopoiesis. Mitochondria are dynamic organelles that continuously undergo fission and fusion processes to maintain their function, integrity, and activity. Mitochondria could be a hub of various biological processes that underlie cellular and systemic homeostasis. Thus, mitochondrial dysfunction could directly lead to the disruption of cellular homeostasis and the development of various disorders, including cancer. Notably, emerging data have revealed that mitochondria dynamics also primarily affect not only mitochondrial function and activity but also cellular homeostasis, the aging process, and tumorigenesis. Here, by focusing on mitochondrial dynamics, we highlight the current understanding of mitochondrial roles as a pathobiological mediator of myeloid malignancies and aging-related clonal hematopoiesis.

RNAi screening reveals a synthetic chemical-genetic interaction between ATP synthase and PFK1 in cancer cells.

To meet cellular bioenergetic and biosynthetic demands, cancer cells remodel their metabolism to increase glycolytic flux, a phenomenon known as the Warburg effect and believed to contribute to cancer malignancy. Among glycolytic enzymes, phosphofructokinase-1 (PFK1) has been shown to act as a rate-limiting enzyme and to facilitate the Warburg effect in cancer cells. In this study, however, we found that decreased PFK1 activity did not affect cell survival or proliferation in cancer cells. This raised a question regarding the importance of PFK1 in malignancy. To gain insights into the role of PFK1 in cancer metabolism and the possibility of adopting it as a novel anticancer therapeutic target, we screened for genes that caused lethality when they were knocked down in the presence of tryptolinamide (TLAM), a PFK1 inhibitor. The screen revealed a synthetic chemical-genetic interaction between genes encoding subunits of ATP synthase (complex V) and TLAM. Indeed, after TLAM treatment, the sensitivity of HeLa cells to oligomycin A (OMA), an ATP synthase inhibitor, was 13,000 times higher than that of untreated cells. Furthermore, this sensitivity potentiation by TLAM treatment was recapitulated by genetic mutations of PFK1. By contrast, TLAM did not potentiate the sensitivity of normal fibroblast cell lines to OMA, possibly due to their reduced energy demands compared to cancer cells. We also showed that the PFK1-mediated glycolytic pathway can act as an energy reservoir. Selective potentiation of the efficacy of ATP synthase inhibitors by PFK1 inhibition may serve as a foundation for novel anticancer therapeutic strategies.

[Novel germline SAMD9 mutation in an elderly patient with myelodysplastic syndrome].

An 80-year-old Japanese male patient presented to our hospital with complaints of fatigue. His peripheral blood tests revealed pancytopenia with predominant lymphocytes and without blasts. The bone marrow (BM) aspiration was unsuccessful due to a dry tap, and the subsequent BM biopsy revealed hypocellular marrow with fibrosis. He was diagnosed with myelodysplastic syndrome (MDS) with excess blasts (EB)-2 based on CD34-positive cells. The chromosome analysis of the BM revealed monosomy 7, and the SAMD9 W22* mutation was detected (variant allele frequency [VAF] of 51.22%) using next-generation sequencing. An identical mutation was observed in the buccal mucosa (VAF of 50%), which was confirmed as a germline mutation. The SAMD9 gene mutation is reported as one of the causative genes for MIRAGE syndrome and child-onset MDS. The present case was considered a loss-of-function mutation due to the near full-length SAMD9 deletion. This is the first adult case of MDS with SAMD9 W22* as a germline mutation.

[A favorable clinical course of acute myeloid leukemia with t (6;21;8)(p23;q22;q22)].

Variants of the t (8;21) (q22;q22) involving chromosome 8, 21, and other chromosomes account for about 3% of all t (8;21) (q22;q22) in patients with acute myeloid leukemia (AML). However, the prognosis of AML with variant t (8;21) remains unknown due to the scarcity of reported cases. Herein we report a case of AML with t (6;21;8) (p23;q22;q22). Fluorescence in situ hybridization confirmed a RUNX1-RUNX1T1 fusion signal on the derivative chromosome 8. This is the first report on a variant of t (8;21) involving the breakpoint 6p23. After induction chemotherapy, our patient achieved complete remission and has been stable for four years.

[Klinefelter's syndrome diagnosed at the onset of acute myeloid leukemia with inv (16) following treatment for germ cell tumor].

A 22-year-old man with a history of mediastinal germ cell tumor, which was diagnosed at age 20 and remained disease-free after chemotherapy, was diagnosed with acute myeloid leukemia (AML) M2 in January 2020. Karyotype analysis of bone marrow (BM) specimen at diagnosis detected 47,XXY, inv (16) in all cells. Following induction treatment, he achieved complete remission with a remarkable decrease in the minimal residual disease marker. Although considered related to therapy, the AML had a prognostically favorable karyotype, and the initial treatment response was very good. He had no human leukocyte antigen-matched sibling donor candidate. Thus, allogeneic hematopoietic stem cell transplantation was not scheduled at the first complete remission. After three cycles of consolidation therapy, he remained disease-free for over one year. Karyotype analysis of BM during remission revealed that all analyzed cells harbored 47,XXY, and Klinefelter syndrome (KS) was diagnosed. Although the patient experienced an adjustment disorder on KS diagnosis, he had overcome the difficulty with the assistance of psycho-oncologists, clinical psychologists, and genetic counselors. Herein, we report this rare case of KS that manifested after AML diagnosis following mediastinal germ cell tumor treatment.

Successful Cord Blood Transplantation for Idiopathic CD4+ Lymphocytopenia.

Idiopathic CD4+ lymphocytopenia (ICL) is the depletion of CD4+ lymphocytes to <300 cells/mm3 without human immunodeficiency virus infection or other causes of lymphocytopenia. ICL causes fatal infections; its etiology remains unclear and it lacks consensus regarding therapeutic options. We report the first patient with ICL who had a successful clinical course following a cord blood transplant (CBT). A 45-year-old woman was diagnosed with ICL and underwent partial hepatectomy for an abscess caused by the Mycobacterium avium complex. No specific gene alterations were detected through next generation sequencing-based evaluation. Following a reduced-intensity conditioning (RIC) regimen consisting of fludarabine, busulfan, and 4 Gy total body irradiation, a single-unit CBT was performed. Neutrophils were engrafted on day +14. CD4+ lymphocyte counts increased to over 300 cells/mm3 on day +436. After 75 months, she was alive without any sequelae. CBT with an RIC regimen could be a curable treatment option for ICL.

Gene rearrangements of MLL and RUNX1 sporadically occur in normal CD34+ cells under cytokine stimulation.

Gene rearrangements of MLL/KMT2A or RUNX1 are the major cause of therapy-related leukemia. Moreover, MLL rearrangements are the major cause of infant leukemia, and RUNX1 rearrangements are frequently detected in cord blood. These genes are sensitive to topoisomerase II inhibitors, and various genes have been identified as potential fusion partners. However, fetal exposure to these inhibitors is rare. Therefore, we postulated that even a proliferation signal itself might induce gene rearrangements in hematopoietic stem cells. To test this hypothesis, we detected gene rearrangements in etoposide-treated or non-treated CD34+ cells cultured with cytokines using inverse PCR. In the etoposide-treated cells, variable-sized rearrangement bands were detected in the RUNX1 and MLL genes at 3 hours of culture, which decreased after 7 days. However, more rearrangement bands were detected in the non-treated cells at 7 days of culture. Such gene rearrangements were also detected in peripheral blood stem cells mobilized by cytokines for transplantation. However, none of these rearranged genes encoded the leukemogenic oncogene, and the cells with rearrangements did not expand. These findings suggest that MLL and RUNX1 rearrangements, which occur with very low frequency in normal hematopoietic progenitor cells, may be induced under cytokine stimulation. Most of the cells with gene rearrangements are likely eliminated, except for leukemia-associated gene rearrangements, resulting in the low prevalence of leukemia development.

Hypoxia/pseudohypoxia-mediated activation of hypoxia-inducible factor-1α in cancer.

Since the first identification of hypoxic cells in sections of carcinomas in the 1950s, hypoxia has been known as a central hallmark of cancer cells and their microenvironment. Indeed, hypoxia benefits cancer cells in their growth, survival, and metastasis. The historical discovery of hypoxia-inducible factor-1α (HIF1A) in the early 1990s had a great influence on the field as many phenomena in hypoxia could be explained by HIF1A. However, not all regions or types of tumors are necessarily hypoxic. Thus, it is difficult to explain whole cancer pathobiology by hypoxia, especially in the early stage of cancer. Upregulation of glucose metabolism in cancer cells has been well known. Oxygen-independent glycolysis is activated in cancer cells even in the normoxia condition, which is known as the Warburg effect. Accumulating evidence and recent advances in cancer metabolism research suggest that hypoxia-independent mechanisms for HIF signaling activation is a hallmark for cancer. There are various mechanisms that generate pseudohypoxic conditions, even in normoxia. Given the importance of HIF1A for cancer pathobiology, the pseudohypoxia concept could shed light on the longstanding mystery of the Warburg effect and accelerate better understanding of the diverse phenomena seen in a variety of cancers.

MDS: Recent progress in molecular pathogenesis and clinical aspects.

Myelodysplastic syndromes (MDS) are defined as hematopoietic stem cell disorders caused by various gene abnormalities. Recent analysis using next generation sequencing has provided great progress in identifying relationships between gene mutations and clinical phenotypes of MDS. It is estimated that one or more gene mutations occur in greater than 90% of MDS patients. More than 50 gene mutations affecting RNA splicing machinery, DNA methylation, histone modifications, transcription factors, signal transduction proteins, and components of the cohesion complex participate in the pathogenesis of MDS. The sequential accumulation of additional cooperating mutations drives disease evolution from clonal hematopoiesis of indeterminate potential (CHIP) to symptomatic MDS and from MDS to acute myelogenous leukemia (AML). Mutations in RNA splicing and DNA methylation occur early and are considered founding mutations, whereas others that occur later are regarded as subclonal mutations. RUNX1 mutations are more likely to be subclonal; however, they apparently play a pivotal role in familial MDS. In addition, large alterations of chromosomes are involved in the pathogenesis of MDS. 5q- syndrome, which leads to haploinsufficiency of the located genes, has consistent clinical features. Understanding gene abnormalities of MDS patients can provide clinical information, including diagnosis, prognostic score, and prediction of response to therapy.

Hmga2 is a direct target gene of RUNX1 and regulates expansion of myeloid progenitors in mice.

RUNX1 is a master transcription factor in hematopoiesis and mediates the specification and homeostasis of hematopoietic stem and progenitor cells (HSPCs). Disruptions in RUNX1 are well known to lead to hematologic disease. In this study, we sought to identify and characterize RUNX1 target genes in HSPCs by performing RUNX1 chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) using a murine HSPC line and complementing this data with our previously described gene expression profiling of primary wild-type and RUNX1-deficient HSPCs (Lineage(-)/cKit(+)/Sca1(+)). From this analysis, we identified and confirmed that Hmga2, a known oncogene, as a direct target of RUNX1. Hmga2 was strongly upregulated in RUNX1-deficient HSPCs, and the promoter of Hmga2 was responsive in a cell-type dependent manner upon coexpression of RUNX1. Conditional Runx1 knockout mice exhibit expansion of their HSPCs and myeloid progenitors as hallmark phenotypes. To further validate and establish that Hmga2 plays a role in inducing HSPC expansion, we generated mouse models of HMGA2 and RUNX1 deficiency. Although mice lacking both factors continued to display higher frequencies of HSPCs, the expansion of myeloid progenitors was effectively rescued. The data presented here establish Hmga2 as a transcriptional target of RUNX1 and a critical regulator of myeloid progenitor expansion.

Hes1 promotes blast crisis in chronic myelogenous leukemia through MMP-9 upregulation in leukemic cells.

High levels of HES1 expression are frequently found in BCR-ABL(+) chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC-like disease; however, the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-κB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of HES1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area-forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, CMPs expressing BCR-ABL and Hes1 secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC-like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells.

[Chronic myelomonocytic leukemia (CMML): recent advances in molecular pathogenesis and treatment].

Chronic myelomonocytic leukemia (CMML) is one of the clonal myeloid neoplasms characterized by persistent monocytosis and dysplasia of myeloid lineage cells. Thus, CMML includes characteristics of both myelodysplastic syndromes and myeloproliferative neoplasms. Clinical features of CMML are quite heterogeneous. There are no disease-specific gene mutations although more than 90% of CMML patients have one or more gene mutations, and most mutations detected in CMML are also seen in other myeloid malignancies. Among these mutations, ASXL1 mutations negatively affect the disease outcome. Moreover, it has been clarified that the clonal architecture of CMML is characterized by linear accumulation of mutations. Recently, international consortium perspectives in diagnostic recommendations and response criteria were published, and clinical reports on CMML, including a new diagnostic method, molecularly integrated CMML-specific prognostic models and therapeutic trials, are increasing. However, despite the existence of several prognostic models of CMML, formal guidelines for the management of CMML are still lacking. An international consortium proposal of uniform guidelines for management of CMML based on a uniform prognostic scoring system is eagerly awaited.

[Chronic Myelomonocytic Leukemia with Myelofibrosis Resulting in Sudden Massive Pleural Effusion during Cytoreductive Therapy with Hydroxycarbamide].

Pleural effusion may occur as a rare complication associated with myeloid hematological malignancies. However, it occasionally occurs in patients with myelodysplastic/myeloproliferative neoplasms(MDS/MPN), especially in chronic myelomonocytic leukemia(CMML)with marked leukocytosis. Pleural effusion can also develop in hematological disorders with bone marrow fibrosis. Here, we report a case of CMML with bone marrow fibrosis, in which massive pleural effusion developed rapidly during cytoreductive therapy with hydroxycarbamide(HU). At the same time, the patient's leukocytosis was well controlled by the HU treatment. Although the cause of the patient's pleural effusion was unclear, despite a detailed thoracoscopic investigation, it is suspected that the invasion of leukemia cells or extramedullary hematopoiesis in the thoracic cavity may have led to this complication. Our findings suggest that in MPN and hematological disorders with bone marrow fibrosis, pleural effusion should be considered as a possible complication and should be carefully monitored, even when cytoreductive therapy is effective.

Long noncoding RNA, CCDC26, controls myeloid leukemia cell growth through regulation of KIT expression.

BACKGROUND: Accumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases, such as cancer. The lncRNA, CCDC26, is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients. RESULTS: We found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26, gene knockdown (KD) was performed using short hairpin RNAs (shRNAs), and four KD clones, in which CCDC26 expression was suppressed to 1% of its normal level, were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA), indicating that transcriptional gene suppression (TGS), not post-transcriptional suppression, was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant, which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast, enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor, KIT, hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03, a KIT-specific inhibitor, eliminated the increased survival of KD clones in the absence of serum. CONCLUSIONS: We suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered.

Recent advances in myelodysplastic syndromes: Molecular pathogenesis and its implications for targeted therapies.

Myelodysplastic syndromes (MDS) are defined as stem cell disorders caused by various gene abnormalities. Recent analysis using next-generation sequencing has provided great advances in identifying relationships between gene mutations and clinical phenotypes of MDS. Gene mutations affecting RNA splicing machinery, DNA methylation, histone modifications, transcription factors, signal transduction proteins and components of the cohesion complex participate in the pathogenesis and progression of MDS. Mutations in RNA splicing and DNA methylation occur early and are considered "founding mutations", whereas others that occur later are regarded as "subclonal mutations". RUNX1 mutations are more likely to subclonal; however, they apparently play a pivotal role in familial MDS. These genetic findings may lead to future therapies for MDS.

RUNX1/AML1 mutant collaborates with BMI1 overexpression in the development of human and murine myelodysplastic syndromes.

RUNX1/AML1 mutations have been identified in myelodysplastic syndromes (MDSs). In a mouse bone marrow transplantation model, a RUNX1 mutant, D171N, was shown to collaborate with Evi1 in the development of MDSs; however, this is rare in humans. Using enforced expression in human CD34(+) cells, we showed that the D171N mutant, the most frequent target of mutation in the RUNX1 gene, had an increased self-renewal capacity, blocked differentiation, dysplasia in all 3 lineages, and tendency for immaturity, but no proliferation ability. BMI1 overexpression was observed in CD34(+) cells from the majority of MDS patients with RUNX1 mutations, but not in D171N-transduced human CD34(+) cells. Cotransduction of D171N and BMI1 demonstrated that BMI1 overexpression conferred proliferation ability to D171N-transduced cells in both human CD34(+) cells and a mouse bone marrow transplantation model. Stepwise transduction of D171N followed by BMI1 in human CD34(+) cells resulted in long-term proliferation with a retained CD34(+) cell fraction, which is quite similar to the phenotype in patients with higher-risk MDSs. Our results indicate that BMI1 overexpression is one of the second hit partner genes of RUNX1 mutations that contribute to the development of MDSs.

Azacitidine and gemtuzumab ozogamicin as post-transplant maintenance therapy for high-risk hematologic malignancies.

Disease recurrence remains the principal cause of treatment failure after allogeneic hematopoietic stem cell transplantation. Post-transplant maintenance therapy with azacitidine (AZA) is promising to prevent relapse but the outcomes are unsatisfactory in patients at high risk of recurrence. Herein, we evaluated the outcome in patients who received AZA and gemtuzumab ozogamicin (GO), anti-CD33 antibody-calicheamicin conjugate, as post-transplant maintenance therapy. Twenty-eight patients with high-risk hematologic malignancies harboring CD33-positive leukemic blasts received the maintenance therapy. AZA (30 mg/m2) was administered for 7 days, followed by GO (3 mg/m2) on day 8. The maximum number of cycles was 4. At transplant, 21 patients (75.0%) had active disease. Their 2-year overall survival, disease-free survival, relapse, and non-relapse mortality rates were 53.6%, 39.3%, 50.0%, and 10.7%, respectively. Of these patients, those with minimal residual disease at the start of maintenance therapy (n = 9) had a higher recurrence rate (66.7% vs. 42.1% at 2 years, P = 0.069) and shorter disease-free survival (11.1% vs. 52.6% at 2 years, P = 0.003). Post-transplant maintenance therapy with AZA and GO was generally tolerable but more than half of the patients eventually relapsed. Further improvements are needed to prevent relapse after transplantation in patients with high-risk hematologic malignancies.

A MAP1B-cortactin-Tks5 axis regulates TNBC invasion and tumorigenesis.

The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.