HOIL-1L deficiency induces cell cycle alteration which causes immaturity of skeletal muscle and cardiomyocytes.

HOIL-1L deficiency was recently reported to be one of the causes of myopathy and dilated cardiomyopathy (DCM). However, the mechanisms by which myopathy and DCM develop have not been clearly elucidated. Here, we sought to elucidate these mechanisms using the murine myoblast cell line C2C12 and disease-specific human induced pluripotent stem cells (hiPSCs). Myotubes differentiated from HOIL-1L-KO C2C12 cells exhibited deteriorated differentiation and mitotic cell accumulation. CMs differentiated from patient-derived hiPSCs had an abnormal morphology with a larger size and were excessively multinucleated compared with CMs differentiated from control hiPSCs. Further analysis of hiPSC-derived CMs showed that HOIL-1L deficiency caused cell cycle alteration and mitotic cell accumulation. These results demonstrate that abnormal cell maturation possibly contribute to the development of myopathy and DCM. In conclusion, HOIL-1L is an important intrinsic regulator of cell cycle-related myotube and CM maturation and cell proliferation.

Efficacy of off-the-shelf bone marrow mesenchymal stem cells for pediatric steroid-refractory acute graft-versus-host disease.

Introduction: Temcell is a mesenchymal stem cell (MSC) product approved for steroid-refractory acute graft-versus-host disease (SR-aGVHD) in Japan. However, reports regarding Temcell's efficacy in pediatric patients have been scarce, and the appropriate use of MSC therapy against pediatric SR-aGVHD also remains to be determined. Patients and Methods: We retrospectively assessed a cohort of pediatric patients treated with Temcell for SR-aGVHD following allogeneic hematopoietic transplantation. MSCs were infused intravenously at a dose of 2 × 106 cells/kg according to the manufacturer's instructions. Results: Twelve patients received eighteen cycles of MSC therapy (median age, 10.3 [1.7-17.8] years), with four receiving additional cycles (one cycle: n = 3, three cycles: n = 1). The severity of aGVHD before MSC therapy was grade I-II in three patients and grade III-IV in nine patients (gut stage 3-4, n= 7; liver stage 3-4; n =2). The median number of immunosuppressive therapy regimens received prior to MSC administration was two (range: 1-5). The first MSC cycle displayed the best overall response rate of 83%, including six patients with a complete response (CR) and with a 49% reduction in the mean daily dose of prednisone after eight weeks. The median time to first response was 3.5 days (range: 2-15 days). Two of the four patients who were re-administered MSCs for recurrent or persistent GVHD achieved a CR. The three-year overall survival rate was 69.4%, while the three-year failure free survival (FFS) rate was 22.2%, with a median FFS of 4.9 months. There were no observable side effects of MSC therapy. Conclusions: MSC therapy appears to be an effective and safe treatment for pediatric SR-aGVHD, with a steroid-sparing effect and satisfactory efficacy upon re-administration. Further studies are needed to determine its appropriate combination with additional treatments and the optimal use of re-administration of MSCs.

Early chemotherapeutic intervention to avoid thyroidectomy in pediatric Langerhans cell histiocytosis with thyroid involvement accompanying tracheal stenosis: a report of two cases.

Thyroid involvement is rare in pediatric Langerhans cell histiocytosis (LCH). It may cause airway narrowing, leading to acute-onset respiratory distress. Severe cases may require emergent surgical interventions such as thyroidectomy, which should be avoided in children due to higher rates of complication, particularly in infancy. There is currently no consensus on the indications for surgical treatment in LCH with thyroid involvement. In this report, we describe the cases of two children who presented with tracheal stenosis caused by thyroid LCH, both of which were successfully treated by early induction of chemotherapy, and one of which was also treated for a shorter duration. Mutation analysis detected in-frame deletions of BRAF exon 12 in both cases. These cases suggest that timely diagnosis and administration of chemotherapy may alleviate severe airway obstruction and reduce the need for thyroidectomy in pediatric patients with thyroid LCH.

RNA-seq-based miRNA signature as an independent predictor of relapse in pediatric B-cell acute lymphoblastic leukemia.

ABSTRACT: Aberrant micro-RNA (miRNA) expression profiles have been associated with disease progression and clinical outcome in pediatric cancers. However, few studies have analyzed genome-wide dysregulation of miRNAs and messenger RNAs (mRNAs) in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To identify novel prognostic factors, we comprehensively investigated miRNA and mRNA sequencing (miRNA-seq and mRNA-seq) data in pediatric BCP-ALL samples with poor outcome. We analyzed 180 patients, including 43 matched pairs at diagnosis and relapse. Consensus clustering of miRNA expression data revealed a distinct profile characterized by mainly downregulation of miRNAs (referred to as an miR-low cluster [MLC]). The MLC profile was not associated with any known genetic subgroups. Intriguingly, patients classified as MLC had significantly shorter event-free survival (median 21 vs 33 months; log-rank P = 3 ×10-5). Furthermore, this poor prognosis was retained even in hyperdiploid ALL. This poor prognostic MLC profiling was confirmed in the validation cohort. Notably, non-MLC profiling at diagnosis (n = 9 of 23; Fisher exact test, P = .039) often changed into MLC profiling at relapse for the same patient. Integrated analysis of miRNA-seq and mRNA-seq data revealed that the transcriptional profile of MLC was characterized by enrichment of MYC target and oxidative phosphorylation genes, reduced intron retention, and low expression of DICER1. Thus, our miRNA-mRNA integration approach yielded a truly unbiased molecular stratification of pediatric BCP-ALL cases based on a novel prognostic miRNA signature, which may lead to better clinical outcomes.

High-dose carboplatin-irinotecan-temozolomide is an effective salvage chemotherapy for relapsed or refractory neuroblastoma.

There is no clear consensus on the most effective treatment for relapsed/refractory high-risk neuroblastoma (NB). We retrospectively assessed seven NB patients with relapsed/refractory disease who received high-dose carboplatin-irinotecan-temozolomide (HD-CIT). Five of seven patients showed favorable therapeutic response (complete remission or partial remission). Regarding toxicity, the cytopenia period tended to prolong when more than three cycles were repeated, but nonhematological toxicities were controllable with general supportive care. Due to its antitumor efficacy and well-tolerated nonhematologic toxicity, HD-CIT is a promising salvage chemotherapy for relapsed/refractory NB. However, it is important to pay attention to the exacerbation of hematological toxicity when repeating the regimen.

Long-term efficacy and safety profile of splenectomy for pediatric chronic immune thrombocytopenia.

There are few reports of the long-term efficacy of splenectomy in children with immune thrombocytopenia (ITP). In a 33-year period, we performed splenectomies in 23 pediatric patients with ITP at a single institution in Japan. The age at surgery was 5-22 years with a median of 10 years. The follow-up period was 1-141 months with a median of 48 months. Before surgery, we confirmed the presence or absence of the accessory spleen by contrast-enhanced CT scan and we recommended vaccination with pneumococcal vaccine. Four patients underwent laparotomy before 1998, and 19 patients underwent laparoscopic surgery after 1999. Splenectomy showed high efficacy with a partial response rate of 83% and a complete response rate of 74%. Complete response was maintained in 70% of patients until the end of the observation period, and 91% were able to discontinue long-term management drugs such as steroids. No serious complications such as infectious diseases were observed. Although the number of cases here was small, the long-term efficacy and safety of splenectomy makes it a viable option in pediatric ITP despite the existence of newer therapeutic agents. Further research is necessary to compare the long-term efficacy and safety of splenectomy with new therapeutic agents.

Use of Cabozantinib to Treat MET -amplified Pediatric Colorectal Cancer.

Pediatric colorectal cancer (CRC) is extremely rare, with little information about genetic profiles compared with adult CRC. Here, a 13-year-old male with advanced CRC underwent cancer gene panel testing, which detected 4 genetic abnormalities ( MET amplification in addition to TP53 , SMAD4 , and CTNNA1 mutations) that might be associated with a poor prognosis. Based on high-level MET amplification, he received a multikinase inhibitor, cabozantinib, after failure of first-line and second-line chemotherapy, resulting in transient disease stabilization. Tailored targeted therapy based on molecular profiling can be an effective treatment strategy for rare cancers such as pediatric CRC.

RUNX1-Survivin Axis Is a Novel Therapeutic Target for Malignant Rhabdoid Tumors.

Malignant rhabdoid tumor (MRT) is a highly aggressive pediatric malignancy with no effective therapy. Therefore, it is necessary to identify a target for the development of novel molecule-targeting therapeutic agents. In this study, we report the importance of the runt-related transcription factor 1 (RUNX1) and RUNX1-Baculoviral IAP (inhibitor of apoptosis) Repeat-Containing 5 (BIRC5/survivin) axis in the proliferation of MRT cells, as it can be used as an ideal target for anti-tumor strategies. The mechanism of this reaction can be explained by the interaction of RUNX1 with the RUNX1-binding DNA sequence located in the survivin promoter and its positive regulation. Specific knockdown of RUNX1 led to decreased expression of survivin, which subsequently suppressed the proliferation of MRT cells in vitro and in vivo. We also found that our novel RUNX inhibitor, Chb-M, which switches off RUNX1 using alkylating agent-conjugated pyrrole-imidazole polyamides designed to specifically bind to consensus RUNX-binding sequences (5'-TGTGGT-3'), inhibited survivin expression in vivo. Taken together, we identified a novel interaction between RUNX1 and survivin in MRT. Therefore the negative regulation of RUNX1 activity may be a novel strategy for MRT treatment.

A RUNX-targeted gene switch-off approach modulates the BIRC5/PIF1-p21 pathway and reduces glioblastoma growth in mice.

Glioblastoma is the most common adult brain tumour, representing a high degree of malignancy. Transcription factors such as RUNX1 are believed to be involved in the malignancy of glioblastoma. RUNX1 functions as an oncogene or tumour suppressor gene with diverse target genes. Details of the effects of RUNX1 on the acquisition of malignancy in glioblastoma remain unclear. Here, we show that RUNX1 downregulates p21 by enhancing expressions of BIRC5 and PIF1, conferring anti-apoptotic properties on glioblastoma. A gene switch-off therapy using alkylating agent-conjugated pyrrole-imidazole polyamides, designed to fit the RUNX1 DNA groove, decreased expression levels of BIRC5 and PIF1 and induced apoptosis and cell cycle arrest via p21. The RUNX1-BIRC5/PIF1-p21 pathway appears to reflect refractory characteristics of glioblastoma and thus holds promise as a therapeutic target. RUNX gene switch-off therapy may represent a novel treatment for glioblastoma.

Chlorambucil-conjugated PI-polyamides (Chb-M'), a transcription inhibitor of RUNX family, has an anti-tumor activity against SHH-type medulloblastoma with p53 mutation.

Malignancy of medulloblastoma depends on its molecular classification. Sonic Hedgehog (SHH)-type medulloblastoma with p53 mutation was recognized as one of the most aggressive types of tumors. We developed a novel drug, chlorambucil-conjugated PI-polyamides (Chb-M'), which was designed to compete with the RUNX consensus DNA-binding site. Chb-M' specifically recognizes this consensus sequence and alkylates it to inhibit the RUNX transcriptional activity. In-silico analysis showed all the RUNX families were upregulated in the SHH-type medulloblastoma. Thus, we tested the anti-tumor effects of Chb-M' in vitro and in vivo using Daoy cell lines, which belong to SHH with p53 mutation. Chb-M' inhibited tumor growth of Daoy cells by inducing apoptosis. The same inhibitory effect was also observed by knocking down of RUNX1 or RUNX2, but not RUNX3. Apoptosis array analysis showed that Chb-M' treatment induced phosphorylation of p53 serine 15 residues. In a subcutaneous tumor model, intratumoral injection of Chb-M' induced tumor growth retardation. Chb-M' mediated inhibition of RUNX1 and RUNX2 can be a novel therapeutic strategy for SHH-type medulloblastoma with p53 mutation.

The first Japanese biobank of patient-derived pediatric acute lymphoblastic leukemia xenograft models.

A lack of practical resources in Japan has limited preclinical discovery and testing of therapies for pediatric relapsed and refractory acute lymphoblastic leukemia (ALL), which has poor outcomes. Here, we established 57 patient-derived xenografts (PDXs) in NOD.Cg-Prkdcscid ll2rgtm1Sug /ShiJic (NOG) mice and created a biobank by preserving PDX cells including three extramedullary relapsed ALL PDXs. We demonstrated that our PDX mice and PDX cells mimicked the biological features of relapsed ALL and that PDX models reproduced treatment-mediated clonal selection. Our PDX biobank is a useful scientific resource for capturing drug sensitivity features of pediatric patients with ALL, providing an essential tool for the development of targeted therapies.

PAX5 alterations in an infant case of KMT2A-rearranged leukemia with lineage switch.

Lineage switch is a rare event at leukemic relapse. While mostly known to occur in KMT2A-rearranged infant leukemia, the underlying mechanism is yet to be depicted. This case report describes a female infant who achieved remission of KMT2A-MLLT3-rearranged acute monocytic leukemia, but 6 months thereafter, relapsed as KMT2A-MLLT3-rearranged acute lymphocytic leukemia. Whole exome sequencing of the bone marrow obtained pre-post lineage switch revealed two somatic mutations of PAX5 in the relapse sample. These two PAX5 alterations were suggested to be loss of function, thus to have played the driver role in the lineage switch from acute monocytic leukemia to acute lymphocytic leukemia.

Alteration of the immune environment in bone marrow from children with recurrent B cell precursor acute lymphoblastic leukemia.

Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High-dimensional single-cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1-polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune-related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1-polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse.

RUNX1 transactivates BCR-ABL1 expression in Philadelphia chromosome positive acute lymphoblastic leukemia.

The emergence of tyrosine kinase inhibitors as part of a front-line treatment has greatly improved the clinical outcome of the patients with Ph+ acute lymphoblastic leukemia (ALL). However, a portion of them still become refractory to the therapy mainly through acquiring mutations in the BCR-ABL1 gene, necessitating a novel strategy to treat tyrosine kinase inhibitor (TKI)-resistant Ph+ ALL cases. In this report, we show evidence that RUNX1 transcription factor stringently controls the expression of BCR-ABL1, which can strategically be targeted by our novel RUNX inhibitor, Chb-M'. Through a series of in vitro experiments, we identified that RUNX1 binds to the promoter of BCR and directly transactivates BCR-ABL1 expression in Ph+ ALL cell lines. These cells showed significantly reduced expression of BCR-ABL1 with suppressed proliferation upon RUNX1 knockdown. Moreover, treatment with Chb-M' consistently downregulated the expression of BCR-ABL1 in these cells and this drug was highly effective even in an imatinib-resistant Ph+ ALL cell line. In good agreement with these findings, forced expression of BCR-ABL1 in these cells conferred relative resistance to Chb-M'. In addition, in vivo experiments with the Ph+ ALL patient-derived xenograft cells showed similar results. In summary, targeting RUNX1 therapeutically in Ph+ ALL cells may lead to overcoming TKI resistance through the transcriptional regulation of BCR-ABL1. Chb-M' could be a novel drug for patients with TKI-resistant refractory Ph+ ALL.