The Response to Oxidative Damage Correlates with Driver Mutations and Clinical Outcome in Patients with Myelofibrosis

Myelofibrosis (MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and no response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, therefore leading to greater genomic instability and promoting cell transformation. In order to unveil the role of driver mutations in oxidative stress, we assessed ROS levels in CD34+ hematopoietic stem/progenitor cells of MF patients. Our results demonstrated that ROS production in CD34+ cells from CALR-mutated MF patients is far greater compared with patients harboring JAK2 mutation, and this leads to increased oxidative DNA damage. Moreover, CALR-mutant cells show less superoxide dismutase (SOD) antioxidant activity than JAK2-mutated ones. Here, we show that high plasma levels of total antioxidant capacity (TAC) correlate with detrimental clinical features, such as high levels of lactate dehydrogenase (LDH) and circulating CD34+ cells. Moreover, in JAK2-mutated patients, high plasma level of TAC is also associated with a poor overall survival (OS), and multivariate analysis demonstrated that high TAC classification is an independent prognostic factor allowing the identification of patients with inferior OS in both DIPSS lowest and highest categories. Altogether, our data suggest that a different capability to respond to oxidative stress can be one of the mechanisms underlying disease progression of myelofibrosis.

IMPROvE-CED Trial: Intracoronary Autologous CD34+ Cell Therapy for Treatment of Coronary Endothelial Dysfunction in Patients With Angina and Non-Obstructive Coronary Arteries

Background: Coronary endothelial dysfunction (CED) causes angina/ischemia in patients with no-obstructive CAD (NOCAD). Patients with CED have decreased number and function of CD34+ cells involved in normal vascular repair with microcirculatory regenerative potential and paracrine anti-inflammatory effects. We evaluated safety and potential efficacy of intracoronary (IC) autologous CD34+ cell therapy for CED. Methods: Twenty NOCAD patients with invasively-diagnosed CED and persistent angina despite maximally-tolerated medical therapy (MTMT) underwent baseline exercise stress test (EST), GCSF-mediated CD34+ cell-mobilization, leukapheresis, and selective 1x105 CD34+ cells/kg infusion into LAD. Invasive CED evaluation and EST were repeated 6-months after cell infusion. Primary endpoints were safety and effect of IC autologous CD34+ cell therapy on CED at 6-months follow-up. Secondary endpoints were change in CCS angina class, as-needed sublingual nitroglycerin use/day, Seattle Angina Questionnaire (SAQ) scores, and exercise time at 6-months. Change in CED was compared to that of 51 historic-control NOCAD patients treated with MTMT alone. Results: Mean age was 52{plus minus}13 years, 75% women. No death, myocardial infarction, or stroke occurred. IC CD34+ cell infusion improved microvascular CED [% acetylcholine-mediated coronary blood flow increased from 7.2 (-18.0-32.4) to 57.6 (16.3-98.3) %, p=0.014], decreased CCS angina class (3.7{plus minus}0.5 to 1.7{plus minus}0.9, Wilcoxon signed-rank test p=0.00018) and sublingual nitroglycerin use/day [1 (0.4-3.5) to 0 (0-1), Wilcoxon signed-rank test p=0.00047], and improved all SAQ scores with no significant change in exercise time at 6-months follow-up. Historic-control patients had no significant change in CED. Conclusion: A single IC autologous CD34+ cell infusion was safe and may potentially be an effective disease-modifying therapy for microvascular CED in humans. Clinical Trial Registration: NCT03471611

CH02 Peptide Promotes CD34+ Umbilical Cord Blood Hematopoietic Stem Cells Ex Vivo Expansion

Background: Umbilical cord blood (UCB) has been clinically used for human hematopoietic stem cells (HSCs) transplantation. However, limited numbers of the functional UCB-HSCs from single cord blood restricts its application in adults, while most of the strategies for stem cells expansion in vitro are either inefficient or costly. To overcome these obstacles, we evaluated the potential role of our newly identified CH02 peptide in ex vivo culture expansion of CD34+ UCB-HSCs. Methods: Enriched human CD34+ progenitor/stem cells populations were cultured in serum-free medium supplemented with different cytokines combinations for 8 days. These cytokines combinations included various concentration of CH02 peptide or the FLT3 ligand, with a cocktail of several growth factors such as IL-6, SCF and TPO. In addition, the global gene expression profile of the CD34+ cells cultured under different conditions were monitored through RNA-seq experiments. Furthermore, the expanded CD34+ cells were topically transplanted into the dorsal wounds of diabetic mice, and the wound closure was observed to evaluate the pro-repair ability of CH02-cultured CD34+ cells.Results: We herein report that the combination of CH02 peptide and other cytokines under the serum-free medium can effectively expand the CD34+ HSCs into 12-fold within 7 days while maintaining their stem cell properties. Moreover, CH02 peptide increased the anti-inflammatory and growth-promoting capacity of CD34+ cells, and thus accelerating wound healing of diabetic mice via promoting the anti-inflammatory and inhibiting the inflammatory factors.Conclusions: Together, our CH02 peptide demonstrated promising potentials to improve HSCs expansion for clinical application.

Comparison of the Transcriptomic Signatures in Pediatric and Adult CML

Children with chronic myeloid leukemia (CML) tend to present with higher white blood counts and larger spleens than adults with CML, suggesting that the biology of pediatric and adult CML may differ. To investigate whether pediatric and adult CML have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy pediatric and adult CD34+ control cells. Using high-throughput RNA sequencing, we found 567 genes (207 up- and 360 downregulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. Directly comparing pediatric and adult CML CD34+ cells, 398 genes (258 up- and 140 downregulated), including many in the Rho pathway, were differentially expressed in pediatric CML CD34+ cells. Using RT-qPCR to verify differentially expressed genes, VAV2 and ARHGAP27 were significantly upregulated in adult CML CD34+ cells compared to pediatric CML CD34+ cells. NCF1, CYBB, and S100A8 were upregulated in adult CML CD34+ cells but not in pediatric CML CD34+ cells, compared to healthy controls. In contrast, DLC1 was significantly upregulated in pediatric CML CD34+ cells but not in adult CML CD34+ cells, compared to healthy controls. These results demonstrate unique molecular characteristics of pediatric CML, such as dysregulation of the Rho pathway, which may contribute to clinical differences between pediatric and adult patients.

Focal disruption of DNA methylation dynamics at enhancers in IDH-mutant AML cells

Recurrent mutations in IDH1 or IDH2 in acute myeloid leukemia (AML) are associated with increased DNA methylation, but the genome-wide patterns of this hypermethylation phenotype have not been comprehensively studied in AML samples. We analyzed whole-genome bisulfite sequencing data from 15 primary AML samples with IDH1 or IDH2 mutations, which identified ~4000 focal regions that were uniquely hypermethylated in IDHmut samples vs. normal CD34+ cells and other AMLs. These regions had modest hypermethylation in AMLs with biallelic TET2 mutations, and levels of 5-hydroxymethylation that were diminished in IDH and TET-mutant samples, indicating that this hypermethylation results from inhibition of TET-mediated demethylation. Focal hypermethylation in IDHmut AMLs occurred at regions with low methylation in CD34+ cells, implying that DNA methylation and demethylation are active at these loci. AML samples containing IDH and DNMT3AR882 mutations were significantly less hypermethylated, suggesting that IDHmut-associated hypermethylation is mediated by DNMT3A. IDHmut-specific hypermethylation was highly enriched for enhancers that form direct interactions with genes involved in normal hematopoiesis and AML, including MYC and ETV6. These results suggest that focal hypermethylation in IDH-mutant AML occurs by altering the balance between DNA methylation and demethylation, and that disruption of these pathways at enhancers may contribute to AML pathogenesis.

A Randomized Study Comparing the Effects of G-CSF and G-CSF/GM-CSF for the Mobilization of Peripheral Blood Stem Cells by Mitoxantrone and High-Dose Cytarabine Chemotherapy

We investigated the efficiency of mitoxantrone (MIT) and high-dose cytarabine (Ara-C) chemotherapy followed by G-CSF and G-CSF/GM-CSF treatments for the mobilization of peripheral blood stem cells (PBSCs) in patients with leukemia and lymphoma. MIT was intravenously injected at 10 mg/(m2·d) for 2 to 3 days, followed by Ara-C injected intravenously at 2 g/m2 every 12 hours for 1 to 2 days. When white blood cell count recovered from the lowest value, 5 to 7.5 μg/ (kg·d) G-CSF was administered in 23 patients for 5 to 7 successive days. Another 27 patients received 3-5 μg/ (kg·d) G-CSF and 3-5μg/ (kg·d) GM-CSF. Autologous peripheral blood mononuclear cells were collected. Levels of CFU-GM and CD34+ cells were determined after unfreezing. The CD34+ cells and CFU-GM yields of 27 patients in G-CSF plus GM-CSF combination group [(8.79±3.11)×106/kg, (3.52±1.34)×105/kg, respectively] were significantly higher than those of patients receiving G-CSF alone (n=23) [(6.14±2.06)×106/kg, (2.03±1.06)×105/kg, respectively (P < 0.05)]. No obvious changes of T lymphocyte subsets in patients were observed when using G-CSF/GM-CSF, but levels of CD34+ cells increased gradually (P>0.05). The end-point separation blood volume was all above trebling TBV. No severe complications were observed during the mobilization and collection. Autologous PBSCT obtained quick hematopoietic reconstitution. In conclusion, MA chemotherapy combined with G-CSF alone and G-CSF/GM-CSF can safely and effectively mobilize autologous PBSCs, while G-CSF plus GM-CSF is superior to G-CSF alone. Large volume leukapheresis is an important method to enhance the production rate of stem cells and decrease harvesting time.