AKT2 inhibition accelerates the acquisition of phagocytic ability in induced pluripotent stem cell-derived neutrophils.

Neutrophils are key components of the immune system that inhibit bacterial infections. Systemic bacterial infections can cause lethal conditions, especially in patients with neutropenia associated with chemotherapy or other systemic illnesses; hence, early detection of the symptoms and prompt management are crucial in such cases. Previously, we established expandable engineered neutrophil-primed progenitors (NeuPs-XL) using human-induced pluripotent stem cells (iPSCs), which can produce neutrophil-like cells at a clinically suitable scale within 4 days of inducing myeloid differentiation. In this study, using small-molecule compound-based screening, we detected that MK-2206, a selective pan-AKT inhibitor, can accelerate this differentiation process, promote phagocytic ability in neutrophils, and enhance cytokine and chemokine expression in response to lipopolysaccharides. The inhibition of AKT2 has been identified as the key mechanism underlying this acceleration. These results can make a substantial contribution to the development of strategies for the prompt production of clinically applicable iPSC-derived neutrophils, which can potentially lead to the management of severe infections associated with life-threatening neutropenia and the effective treatment of related health conditions in the future.

Heterozygous Dnmt3a R878C induces expansion of quiescent hematopoietic stem cell pool.

Somatic mutation of DNMT3A (DNA methyltransferase 3 alpha) is implicated in the development of a wide range of hematological disorders, including clonal hematopoiesis of indeterminate potential. To elucidate the functional roles of endogenous levels of a DNMT3A R882 mutant, we generated a novel Dnmt3a R878C conditional knock-in mouse model. In contrast to viable heterozygotes, mice homozygous for the Dnmt3a R878C mutation in the hematopoietic system were not viable (Dnmt3a R878C is homologous to human DNMT3A R882C). Hematopoietic cell-specific heterozygous expression of Dnmt3a R878C led to significant expansion of adult quiescent hematopoietic stem cells (HSCs); however, these mice had no hematological malignancies. The expanding HSC population in heterozygous Dnmt3a R878C knock-in mice had an accumulation of G0-phase cells. In contrast to aberrantly enhanced self-renewal capacity in vitro, heterozygous Dnmt3a R878C knock-in HSCs had no competitive repopulating advantage in vivo over wild-type HSCs. Considering the capacity of the heterozygous Dnmt3a R878C mutant for HSC pool expansion, our Dnmt3a R878C knock-in mouse line is a useful platform on which to dissect the pathophysiology of clonal hematopoiesis. This mouse line can also help to elucidate the biological and molecular actions of DNMT3A mutations in the malignant transformation of normal HSCs.

CAMK2G is identified as a novel therapeutic target for myelofibrosis.

Although JAK1/2 inhibition is effective in alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here, we established induced pluripotent stem cells (iPSCs) derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were used for chemical screening, and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutation recurrently detected in MF patients, were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 γ subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515L decreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes, such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

Efficient production of human neutrophils from iPSCs that prevent murine lethal infection with immune cell recruitment.

Neutrophils play an essential role in innate immune responses to bacterial and fungal infections, and loss of neutrophil function can increase the risk of acquiring lethal infections in clinical settings. Here, we show that engineered neutrophil-primed progenitors derived from human induced pluripotent stem cells can produce functional neutrophil-like cells at a clinically applicable scale that can act rapidly in vivo against lethal bacterial infections. Using 5 different mouse models, we systematically demonstrated that these neutrophil-like cells migrate to sites of inflammation and infection and increase survival against bacterial infection. In addition, we found that these human neutrophil-like cells can recruit murine immune cells. This system potentially provides a straight-forward solution for patients with neutrophil deficiency: an off-the-shelf neutrophil transfusion. This platform should facilitate the administration of human neutrophils for a broad spectrum of physiological and pathological conditions.

Loss-of-function mutations in BCOR contribute to chemotherapy resistance in acute myeloid leukemia.

Primary refractory acute myeloid leukemia (AML) is unresponsive to conventional chemotherapy and has a poor prognosis. Despite the recent identification of novel driver mutations and advances in the understanding of the molecular pathogenesis, little is known about the relationship between genetic abnormalities and chemoresistance in AML. In this study, we subjected 39 samples from patients with primary refractory AML to whole-exome and targeted sequencing analyses to identify somatic mutations contributing to chemoresistance in AML. First, we identified 49 genes that might contribute to chemotherapy resistance through the whole-exome sequencing of samples from 6 patients with primary refractory AML. We then identified a significantly higher frequency of mutations in the gene encoding BCL-6 co-repressor (BCOR) in patients with primary refractory AML through the targeted sequencing of all coding sequence of 49 genes. Notably, the presence of BCOR mutations appeared to have a negative impact on prognosis in our cohort and previous larger studies. Subsequently, to investigate the biological effect of BCOR mutations on sensitivity to anticancer drugs, we established BCOR knockout human leukemic cell lines using the CRISPR/Cas9 system. Here, BCOR knockout cell lines exhibited statistically significant reductions in sensitivity to anticancer drugs, compared with the wild-type controls both in vitro and in vivo in xenograft mouse models. In conclusion, loss-of-function BCOR mutations appear to contribute to chemotherapy resistance and may be a promising therapeutic target in primary refractory AML.

Difference of preventing effects of G-CSF according to age in patients with malignant lymphoma: A nation-wide analysis in Japan.

INTRODUCTION: There has been no comprehensive analysis of the age-specific efficacy of G-CSF to prevent febrile neutropenia (FN). We evaluated factors associated with FN occurrence according to patient age in rituximab-cyclophosphamide-doxorubicin-vincristine-prednisolone (R-CHOP) treatment. METHODS: We retrospectively reviewed diffuse large B-cell lymphoma (DLBCL) patients aged ≥50 years, who underwent the first R-CHOP cycle between July 2010 and March 2017, using a Japanese inpatient database. Multivariable logistic regression analysis was performed to identify the factors associated with FN. RESULTS: A total of 16,399 patients with untreated DLBCL were identified. Primary prophylaxis with pegfilgrastim was significantly associated with the lower occurrence of FN (odds ratio: 0.71 [95% confidence interval: 0.51-0.99]). Subgroup analysis according to age was then performed. Although there was no significance, primary prophylaxis with pegfilgrastim tended to have a lower odds ratio for the occurrence of FN in patients aged 50-60 years (0.86 [0.39-1.89]) and 61-70 years (0.64 [0.36-1.13]). In patients aged 71-80 years, primary prophylaxis with pegfilgrastim was significantly associated with reduced FN occurrence (0.46 [0.26-0.80]). Notably, in patients aged >80 years, the use of pegfilgrastim tended to be associated with a rather higher occurrence of FN (1.55 [0.84-2.87]). CONCLUSIONS: Preventing effect of G-CSF may be limited in patients aged >80 years.

Primary prophylaxis with pegfilgrastim in patients with newly-diagnosed diffuse large B-cell lymphoma: propensity score and instrumental variable analyses.

The clinical impact of pegfilgrastim in day-to-day practice remains unclear. This study evaluated the effectiveness of pegfilgrastim compared with daily filgrastim in patients with DLBCL who received the first-cycle of R-CHOP treatment by using a Japanese national inpatient database. Patient characteristics were adjusted by using propensity-score matching and stabilized inverse probability of treatment weighting (IPTW). In 1295 propensity-score-matched pairs, the incidence of febrile neutropenia was significantly lower in the pegfilgrastim group (risk difference 6.1%, 95% CI 4.1%-8.1%) than in the filgrastim group. In the pegfilgrastim group, the length of hospital stay and the total costs were also significantly reduced (percent reduction 34% [95% CI: 31%-37%], percent reduction 12% [95% CI: 9%-15%], respectively). The stabilized IPTW showed comparable results. In day-to-day practice, the simple mode of pegfilgrastim administration may be advantageous.

Significance of biopsy with ERCP for diagnosis of bile duct invasion of DLBCL.

Obstructive jaundice is an initial symptom in 1-2% of diffuse large B cell lymphoma (DLBCL) cases. The major cause of bile duct obstruction in patients with DLBCL is extrinsic compression by enlarged lymph nodes. In such cases, the existence of bile duct invasion of lymphoma is rarely mentioned or observed pathologically, so the ratio of bile duct invasion to the total cases of obstructive jaundice, and its significance remains unknown. We report two cases of DLBCL presenting as an obstructive jaundice, in which we demonstrated bile duct invasion pathologically by biopsy from the wall of common bile duct with endoscopic retrograde cholangiopancreatography (ERCP). Endoscopic stent placement is a minimally invasive procedure to relieve cholestasis and is effective for diagnosing bile duct invasion. This procedure should thus be performed in all cases of obstructive jaundice caused by lymphoma to evaluate for bile duct invasion. Our cases suggest that ERCP may be useful as a diagnostic procedure for bile duct invasion.

Arteritis after administration of granulocyte colony-stimulating factor: a case series.

Granulocyte colony-stimulating factor (G-CSF) is commonly administered to prevent serious complications caused by chemotherapy-induced neutropenia; however, several cases of arteritis following the administration of G-CSF have been reported. Here, we report three cases of patients with non-Hodgkin lymphomas (NHLs) who developed arteritis after the administration of G-CSF, estimate the probability of adverse drug reaction caused by G-CSF with two distinct algorithms, and review the literatures. Both algorithms indicated a causal relationship between G-CSF and arteritis. In a literature review of seven reported cases, including our three patients, the time from the administration of G-CSF to the onset of arteritis ranged from 9 days to 6 months, and five patients were treated with steroids. In one of our three cases, a 62-year-old female with NHL developed arteritis twice in different courses of chemotherapy. Hydrocortisone was administered in the second event, leading to prompt relief of the manifestation and abnormal laboratory data. This finding suggests steroids may be effective for arteritis. In conclusion, although the number of reported cases is limited, there appears to be an association between arteritis and the administration of G-CSF, and steroids are an effective therapeutic option.

Using patient-derived iPSCs to develop humanized mouse models for chronic myelomonocytic leukemia and therapeutic drug identification, including liposomal clodronate.

Chronic myelomonocytic leukemia (CMML) is an entity of myelodysplastic syndrome/myeloproliferative neoplasm. Although CMML can be cured with allogeneic stem cell transplantation, its prognosis is generally very poor due to the limited efficacy of chemotherapy and to the patient's age, which is usually not eligible for transplantation. Comprehensive analysis of CMML pathophysiology and the development of therapeutic agents have been limited partly due to the lack of cell lines in CMML and the limited developments of mouse models. After successfully establishing patient's derived disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with CMML, we utilized these CMML-iPSCs to achieve hematopoietic re-differentiation in vitro, created a humanized CMML mouse model via teratomas, and developed a drug-testing system. The clinical characteristics of CMML were recapitulated following hematopoietic re-differentiation in vitro and a humanized CMML mouse model in vivo. The drug-testing system using CMML-iPSCs identified a MEK inhibitor, a Ras inhibitor, and liposomal clodronate as potential drugs for treating CMML. Clodronate is a drug commonly used as a bisphosphonate for osteoporosis. In this study, the liposomalization of clodronate enhanced its effectiveness in these assays, suggesting that this variation of clodronate may be adopted as a repositioned drug for CMML therapy.

ADAM8 Is an Antigen of Tyrosine Kinase Inhibitor-Resistant Chronic Myeloid Leukemia Cells Identified by Patient-Derived Induced Pluripotent Stem Cells.

Properties of cancer stem cells involved in drug resistance and relapse have significant effects on clinical outcome. Although tyrosine kinase inhibitors (TKIs) have dramatically improved survival of patients with chronic myeloid leukemia (CML), TKIs have not fully cured CML due to TKI-resistant CML stem cells. Moreover, relapse after discontinuation of TKIs has not been predicted in CML patients with the best TKI response. In our study, a model of CML stem cells derived from CML induced pluripotent stem cells identified ADAM8 as an antigen of TKI-resistant CML cells. The inhibition of expression or metalloproteinase activity of ADAM8 restored TKI sensitivity in primary samples. In addition, residual CML cells in patients with optimal TKI response were concentrated in the ADAM8+ population. Our study demonstrates that ADAM8 is a marker of residual CML cells even in patients with optimal TKI response and would be a predictor of relapse and a therapeutic target of TKI-resistant CML cells.

Modeling of hematologic malignancies by iPS technology.

Induced pluripotent stem cells (iPSCs) can be generated from various types of cells with transduction of defined transcription factors. Patient-derived iPSCs are becoming commonly utilized for understanding the molecular pathways involved in disease and for the development of novel targeted therapies. With the use of patient-derived iPSCs differentiated to specific-lineage cells, the potency and toxicity of drug candidates can be evaluated. In the past, patient-derived iPSCs were mainly established from patients of inherited hematologic diseases, followed by the expansion of target to acquired diseases like myeloproliferative neoplasms. Thanks to the rapid development of novel genome editing technologies, we can now utilize genetically modified and unprocessed iPSCs more readily than before. These technologies, which enable us to modulate genetic status or even chromosome structure at the right time, could help the elucidation of pathogenesis of hematologic diseases. If iPSC-derived hematopoietic cells are to be robustly reconstituted in vivo as a consequence of the development of reprogramming and conversion technology, research on leukemic stem cells must be widely promoted. Therefore, iPSC technology has great potential on oncology research using patient samples.

Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis.

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.

Pituitary lymphoma developing within pituitary adenoma.

Lymphoma occurring in the pituitary gland is an exceedingly infrequent event. Here, we describe a case of pituitary lymphoma complicating recurrent pituitary adenoma. A 56-year-old male with a history of pituitary adenoma was diagnosed with diffuse large B-cell lymphoma (DLBCL) of the left ocular adnexa, which was successfully treated by standard chemotherapy and local radiotherapy. Eight months later, he complained of diplopia and bitemporal hemianopia. Brain magnetic resonance imaging detected a suprasellar tumor. Transsphenoidal biopsy of the mass was performed, and histopathological examination revealed DLBCL admixed with pituitary adenoma. On a review of the literature, we found that pituitary lymphoma developing within adenoma is a recurrent phenomenon. The composite tumor is likely to be characterized by suprasellar involvement and presentation of visual disturbances. Moreover, in the present case, the suprasellar tumor remained visible after autologous peripheral stem cell transplant, likely due to the residual pituitary adenoma. We therefore recommend that refractory pituitary lymphoma should be vigorously biopsied in search of possibly underlying adenoma.