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BackgroundDermal fibrosis occurs in many human diseases, particularly systemic sclerosis (SSc) where persistent inflammation leads to collagen deposition and fiber formation in skin and multiple organs. The family of protein kinase D (PKD) has been linked to inflammatory responses in various pathological conditions, however, its role in inflammation-induced dermal fibrosis has not been well defined. Here, using a murine fibrosis model that gives rise to dermal lesions similar to those in SSc, we investigated the role of PKD in dermal fibrosis in mice lacking PKD2 activity.MethodsHomozygous kinase-dead PKD2 knock-in mice (PKD2SSAA/SSAA-KI) were obtained through intercrossing mice heterozygous for PKD2S707A/S711A(PKD2SSAA). The wild-type and KI mice were subjected to repeated subcutaneous injection of bleomycin (BLM) to induce dermal inflammation and fibrosis. As controls, mice were injected with PBS. At the end of the experiment, mouse skin at the injection site was dissected, stained, and analyzed for morphological changes and expression of inflammatory and fibrotic markers. PKD-regulated signaling pathways were examined by real-time RT-qPCR and Western blotting. In a separate experiment, BLM-treated mice were administered with or without a PKD inhibitor, CRT0066101 (CRT). The effects of CRT on dermal fibrosis were analyzed similarly. The identity of the PKD expressing cells were probed using myeloid lineage markers CD45, CD68 in BLM-treated mouse tissues.ResultsDermal thickness and collagen fibers of kinase-dead PKD2-KI mice were significantly reduced in response to BLM treatment as compared to the wild-type mice. These mice also exhibited reduced α-smooth muscle actin (α-SMA) and collagen expression. At molecular levels, both transforming growth factor β1 (TGF-β1) and interleukin-6 (IL-6) mRNAs were decreased in the KI mice treated with BLM as compared to those in the wild-type mice. Similarly, CRT significantly blocked BLM-induced dermal fibrosis and inhibited the expression of α-SMA, collagen, and IL-6 expression. Further analysis indicated that PKD2 was mainly expressed in CD45+/CD68+myeloid cells that could be recruited to the lesional sites to promote the fibrotic process of the skin in response to BLM.ConclusionsKnock-in of the kinase-dead PKD2 or inhibition of PKD activity in mice protected against BLM-induced dermal fibrosis by reducing dermis thickness and expression of fibrotic biomarkers including α-SMA, collagen, and inflammatory/fibrotic mediators including TGF-β1 and IL-6. PKD2 does this potentially through modulating the recruitment and function of myeloid cells in skin of BLM-treated mice. Overall, our study demonstrated a potential critical role of PKD catalytic activity in inflammation-induced dermal fibrosis.

Supplemental Digital Content is available in the text., Objective Genetic alterations in CDKN2A tumor suppressor gene on chromosome 9p21 confer a predisposition to childhood acute lymphoblastic leukemia (ALL). Genome-wide association studies have identified missense variants in CDKN2A associated with the development of ALL. This study systematically evaluated the effects of CDKN2A coding variants on ALL risk. Methods We genotyped the CDKN2A coding region in 308 childhood ALL cases enrolled in CCCG-ALL-2015 clinical trials by Sanger Sequencing. Cell growth assay, cell cycle assay, MTT-based cell toxicity assay, and western blot were performed to assess the CDKN2A coding variants on ALL predisposition. Results We identified 10 novel exonic germline variants, including 6 missense mutations (p.A21V, p.G45A and p.V115L of p16INK4A; p.T31R, p.R90G, and p.R129L of p14ARF) and 1 nonsense mutation and 1 heterozygous termination codon mutation in exon 2 (p16INK4A p.S129X). Functional studies indicate that five novel variants resulted in reduced tumor suppressor activity of p16INK4A, and increased the susceptibility to the leukemic transformation of hematopoietic progenitor cells. Compared to other variants, p.H142R contributes higher sensitivity to CDK4/6 inhibitors. Conclusion These findings provide direct insight into the influence of inherited genetic variants at the CDKN2A coding region on the development of ALL and the precise clinical application of CDK4/6 inhibitors.

Through the advancements in recent decades, childhood acute lymphoblastic leukemia (ALL) is gradually becoming a highly curable disease. However, the truth is there remaining relapse in ∼15% of ALL cases with dismal outcomes.

Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with an overall prevalence of 4/100,000, accounting for 25–30% of all childhood cancers. With advances in childhood ALL treatment, the cure rate for childhood ALL has exceeded 80% in most countries. However, refractory/relapsed ALL remains a leading cause of treatment failure and subsequent death. Forkhead box O1 (FOXO1) belongs to the forkhead family of transcription factors, but its role in B-cell ALL (B-ALL) has not been determined yet.Procedures: RNA sequencing was applied to an ALL case with induction failure (IF) to identify the possible genetic events. A cytokine-dependent growth assay in Ba/F3 cells was used to test the leukemic transformation capacity of MEIS1–FOXO1. The propidium iodide (PI) staining method was used to evaluate the effect of MEIS1–FOXO1 on cycle distribution. FOXO1 transactivity was examined using a luciferase reporter assay. FOXO1 mRNA expression levels were examined using real-time quantitative PCR among 40 children with B-ALL treated with the CCCG-ALL-2015 protocol. Association analysis was performed to test the correlation of FOXO1 transcription with childhood B-ALL prognosis and relapse in a series of GEO datasets. An MTT assay was performed to test the drug sensitivity.Results: In this ALL case with IF, we identified a novel MEIS1–FOXO1 fusion gene. The transactivity of MEIS1–FOXO1 was significantly lower than that of wild-type FOXO1. MEIS1–FOXO1 potentiated leukemia transformation and promoted Ba/F3 cell cycle S-phase entry. Low FOXO1 transcription levels were found to be strongly associated with unfavorable ALL subtype, minimal residual disease (MRD) positivity, and relapse. Lower FOXO1 expression was associated with prednisone and cyclophosphamide resistance.Conclusions: Low FOXO1 transcription was associated with high-risk stratification and relapse in children with B-ALL, probably due to multi-drug resistance.

Kankan Gao,1 Xiaoshan Guan,1 Lanlan Zeng,1 Jiabi Qian,2 Sufei Zhu,1 Qiulian Deng,1 Huamin Zhong,1 Shuying Pang,1 Fei Gao,1 Jielin Wang,1 Yan Long,1 Chien-Yi Chang,3 Haiying Liu1 1Clinical Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China; 2Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China; 3School of Chemistry and Biosciences, University of Bradford, Bradford, BD7 1DP, UK Background: A multidrug-resistant (MDR) RR2 gene cluster was identified by whole-genome sequencing in several highly virulent (ST-17) Group B streptococcus (GBS) isolates, which caused neonatal invasive infections in southern China in 2016. Tracing the transmission and distribution of MDR isolates in this area is important for the effective management of future infections. The aim of this study was to obtain longitudinal data of MDR isolates to monitor epidemiological trends of general common isolates in southern China, and provide evidence for future characterization of antimicrobial resistance mechanisms.Methods: Clinical information and antimicrobial susceptibility of GBS isolates were acquired from electronic information management system databases of the hospital under study between January 2011 and December 2017. To confirm the presence of intact RR2, the tetO, ant6, lnuB, and ant9 genes located upstream, midstream, and downstream of RR2 were detected by PCR and DNA sequencing.Results: A total of 149 cases of neonatal invasive GBS infection were identified during the period 2011–2017. Among them, 119 cases (79.9%) were caused by MDR isolates, with a general increasing trend over the past 7 years. Further characterization of 11 isolates showed that six isolates causing late-onset disease (LOD) carry the tetO, ant6, and lnuB genes, which are located on RR2. Moreover, lnuB and ant9 consistently co-occurred in GBS isolates, which suggests their close proximity to one another in the RR2 gene cluster.Conclusion: The MDR GBS is responsible for a large number of neonatal invasive infections and occurs with increasing frequency over time. Particularly, the MDR GBS isolates that cause LOD are more likely to carry the RR2 gene cluster, compared with those that cause early-onset disease. The rise in number of MDR GBS isolates emphasizes the pressing need for continuous surveillance to monitor their antibiotic susceptibility and epidemiology. Keywords: invasive infection, gene cluster, neonates, multidrug-resistance

Figure S6. SCF, CCL5, and CCL11 rescued MCs migration inhibited by CM from PC-3M cells with PKD silencing (PDF 2500 kb)

Figure S5. PKD2/3 silencing of prostate cancer cells reduced angiogenic factor expression in P815 MCs cells. (PDF 785 kb)

Figure S1. mRNA level of PKD1/2/3 expression from prostate cancer TCGA data. (PDF 64 kb)

Figure S3. PKD2/3 in prostate cancer cells promoted chemotactic migration of mast cells. (PDF 1025 kb)

Figure S8. PKD2/3 modulated Erk1/2 and NF-ÎşB activity in prostate cancer cells in response to PMA. (PDF 812 kb)

Figure S4. Effect of PKD2 and PKD3 derived prostate cancer cells on endothelial cells tube formation in vitro. (PDF 832 kb)

Figure S7. PKD2/3 did not interact with p38. (PDF 466 kb)

Supplementary data. (DOCX 21 kb)

Figure S2. Autophosphorylation of PKD at s916 in prostate tissue. (PDF 1121 kb)

Accumulating evidence has confirmed that inherited genetic variations play multi-dimensional roles in childhood acute lymphoblastic leukemia (ALL), i.e., leukemia susceptibility, treatment response, chemotherapy tolerance, and relapse. Germline variants at the GATA3 locus increase the risk of developing Philadelphia chromosome-like ALL (Ph-like ALL) and inferior outcomes in childhood B-ALL among European and American cohorts. However, the role of inherited GATA3 variants in Han Chinese children with ALL still remains unclear. To primarily identify the association of inherited GATA3 variants with treatment response, we retrospectively collected 273 childhood B-ALL blood samples after complete remission was achieved according to the Chinese Childhood Cancer Group ALL 2015. We then genotyped rs3824662 and rs3781093 in the GATA3 locus. The risk allele frequencies of rs3824662 and rs3781093 were 35.7% and 36.3%, respectively, consistent with the 1000 Genomes Project. Using a logistic regression model, we correlated the GATA3 genotype with minimal residual disease (MRD) level. In our single center, we found that GATA3 rs3824662 A allele and rs3781093 C allele statistically associated with positive MRD (the cut-off was >=0.01%, P=0.046 and 0.038, respectively). The A allele in rs3824662 and C allele in rs3781093 linked to 2-fold increase in the risk of MRD compared with their wildtype allele. To explore the biological functions of these two germline SNP variants, we first utilized luciferase reporter assay to determine the impact of GATA3 variants on its transcription activity. Interestingly, the rs3824662 risk A allele significantly increased enhancer activity, while the rs3781093 did not show any effect. We next genetically modified rs3824662 from wild-type C allele to A allele in the lymphoblastoid cell GM12878 using clustered regularly interspaced short palindromic repeats/associated 9 (CRISPR/Cas9) gene editing system. Compared with wildtype GM12878 cells, ~3-folds higher GATA3 transcription level was found in GM12878 cells with A/A or A/C genotype. Integrating the high risk of MRD and upregulated GATA3 expression, we proposed that GATA3 rs3824662 A allele might contribute to poor treatment response by promoting GATA3 transcription. To clarify the association of GATA3 expression with the sensitivity of ALL chemotherapeutic drugs, we retrieved GSE653 and GSE654 expression data for analysis and found that high GATA3 expression significantly correlated with L-asparaginase (L-Asp) and daunorubicin (DNR) resistance. To further confirm the correlation, we ectopically overexpressed GATA3 in B-ALL cell line (Nalm6) and only L-asp resistance was validated. L-asp resistance induced by GATA3 over-expression was rescued by GATA3 interference, consolidating the association between GATA3 and L-asp resistance in B-ALL cells. Next, we probed the mechanism of GATA3-mediated L-asp resistance in B-ALL. We analyzed the association between inherited GATA3 variants and L-asp allergy, and did not identify statistical significance. Meanwhile, we couldn't find the correlation between GATA3 and ASNS, suggesting that ASNS might not be the cause either. Intriguingly, we found GATA3 over-expression induced the activation of autophagy-related genes, BECN1 and ATG5, which was reported to be associated with L-Asp resistance. Tests on primary B-ALL samples further confirmed this findings. To further confirm the effect of GATA3 on autophagy, we cloned highly conserved sequence BECN1 or ATG5 promoter region into luciferase reporter constructs. The Over-expression of GATA3 dramatically increased luciferase activity compared with the corresponding empty vector (P = 0.0098 and 0.0114, respectively), indicating that GATA3 expression were functionally activate their transcription. Taken together, all these data indicated that higher GATA3 expression might induce L-Asp resistance in B-ALL cells via autophagy activation. In conclusion, we first identified that GATA3 rs3824662 associated with the risk of MRD in the childhood ALL cohorts of Han ethnicity. Mechanistic study showed that inherited GATA3 variants possibly contributed to L-Asp resistance via autophagy activation induced by promoting GATA3 enhancer activity, providing new insights into the rationale for the future development of combinational treatment of L-Asp and anti-autophagy regimen in ALL patients. Figure Disclosures No relevant conflicts of interest to declare.

The protein kinase D family of serine/threonine kinases, particularly PKD1, has been implicated in the regulation of a complex array of fundamental biological processes. However, its function and mechanism underlying PKD1-mediated the bone development and osteoblast differentiation are not fully understood. Here we demonstrate that loss of PKD1 function led to impaired bone development and osteoblast differentiation through STAT3 and p38 MAPK signaling using in vitro and in vivo bone-specific conditional PKD1-knockout (PKD1-KO) mice models. These mice developed markedly craniofacial dysplasia, scapula dysplasia, long bone length shortage and body weight decrease compared with wild-type littermates. Moreover, deletion of PKD1 in vivo reduced trabecular development and activity of osteoblast development, confirmed by Micro-CT and histological staining as well as expression of osteoblastic marker (OPN, Runx2 and OSX). Mechanistically, loss of PKD1 mediated the downregulation of osteoblast markers and impaired osteoblast differentiation through STAT3 and p38 MAPK signaling pathways. Taken together, these results demonstrated that PKD1 contributes to the osteoblast differentiation and bone development via elevation of osteoblast markers through activation of STAT3 and p38 MAPK signaling pathways.

Background: Thanks to the total therapy and systemic basic-translation research, the overall survival rate in children with acute lymphoblastic leukemia (ALL) has dramatically improved to almost 90% over these past few decades. FOXO1 gene belongs to the forkhead family of transcription factors, which play roles in myogenic growth and differentiation. Translocation of FOXO1 with PAX3 has been reported in pediatric alveolar rhabdomyosarcoma. In B-cell precursor ALL, two cases with FOXO1 fusions have been identified already, while its function on ALL remains unknown. Here, we report a novel MEIS1-FOXO1 fusion gene in a case with B-ALL. Methods: Flowcytometery, karyotype, RT-PCR and fluorescence in were employed, MEIS1-FOXO1 was identified as novel fusion gene in a case of pediatric BCP-ALL. Using IL-3 dependent BaF3 cells as study model to test the leukemia transformation potential of MEIS1-FOXO1. Results: A novel MEIS1-FOXO1 fusion was identified in one cease of pediatric B-ALL. Panel next generation sequencing (NGS) showed that the leukemia clone had concurrent NRASG12D, TP53R273H, WHSC1E1099K, ABCC1R1166X, PHGR1H37P, HOXA3P219L and DSTP4606L somatic mutation. This patient was enrolled in CCCG-ALL2015 clinical trial (ChiCTR-IPR-14005706) and achieved completed remission and low minimal residual disease (MRD) level (MRD Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background: Increasing evidence has pointed out that inherited NUDT15 variants are strongly associated with 6-mercaptopurine (6-MP) toxicities that may require therapy interruption in diseases such as acute lymphoblastic leukemia (ALL), inflammatory bowel diseases, and rheumatic diseases. However, the consequences and costs incurred from treating 6-MP-induced toxicities due to inherited NUDT15 variants are not well explored so far. Objective: This study aims to retrospectively describe the infection rate and medical costs incurred from the treatment of 6-MP toxicities in the absence of NUDT15 genotype-guided dose adjustment in childhood ALL patients who received systemic therapy in our institution, and to compare infection rate and costs between individuals with and without NUDT15 variants. Methods: The study population involved childhood ALL patients below 14 years of age, who were enrolled in CCCG-ALL-2015 (ChiCTR-IPR-14005706) treatment. Patients were categorized into low-, intermediate- and high-risk based on clinical presentation, cytogenetic abnormalities and therapeutic minimal residual disease level. 6-MP was administered at an initial dose of 50mg/m2, and was modified at subsequent administrations based on presenting toxicities and physicians' clinical discretion. Data on infections, as well as medical costs attributable to 6-MP induced toxicities (infections, rash, mucositis, diarrhea etc.), were retrieved retrospectively and analyzed in chronological phases of treatment based on the protocol. Costs were converted to US dollars. Descriptive statistics was used to summarize all clinical and cost data. Independent t-test was used to compare clinical cost data between patients with and without NUDT15 variants for overall, as well as for each stage of therapy. Results: A total of 144 patients were included in this study, of whom 45 patients (31%) were found to have NUDT15 variants (13 cases with exon 1 variant, 16 cases with concurrence of exon 1 and 3 variants, and 16 cases with exon 3 variants) after retrospective screening. However, we did not identify patients with TPMT susceptible variants in our cohort. Patients with NUDT15 inherited variants were more susceptible to myeloisuppression (Figure 1A, variants with mean ANC [95% CI] 0.21 [0.12-0.29]) vs wild type 0.71 [0.61-.81], P Conclusion: We found that patients with NUDT15 variants demonstrated increased susceptibility to severe infections. They also incurred higher medical costs associated with the management of 6-MP toxicities without prior NUDT15-guided dose adjustment, particularly during the early maintenance phase. These preliminary findings suggest that routine pre-treatment screening for NUDT15 variants and early NUDT15-guided 6-MP dose modification may potentially reduce the incidence and cost of managing 6-MP toxicities. A larger prospective cohort should be conducted to validate our findings and thus benefit the children with ALL. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.