Effect of CD34⁺ Total/Myeloid CD34⁺ Cell Progenitors and B-Lymphoid Progenitors Within the Bone Marrow Grafts on the Hematopoietic Recovery After Hematopoietic Stem Cell Transplantation.

OBJECTIVES: The influence of CD34⁺ cells on transplant outcomes following hematopoietic stem cell transplantation remains controversial. A minimum of 2.0 to 2.5 million CD34⁺ cells/kg of patient weight is requested for a rapid and durable engraftment. The aim of this study was to detect the ratio of CD34+ B-lymphoid progenitors (hematogones) in bone marrow grafts and investigate their effects on hematopoietic recovery after transplant. MATERIALS AND METHODS: Our study included 41 patients who received a bone marrow graft from their HLA-matched donor from 2016 through 2019. The CD34⁺ cell numbers within the graft were detected using Stem-Kit (Beckman Coulter). The ratio of CD34⁺ hematogones was determined either by their light scatter characteristics or by the detection of CD34, CD19, or CD10 coexpressing cells in a separate tube. RESULTS: The median number of CD34⁺ cells was 5.9 × 106/kg (0.8-14.3 × 106/kg). The CD34⁺ cells consisted of 71% (range, 35.7%-100%) and 29% (range, 5.7%-64.3%) myeloid and B-lymphoid progenitors, respectively. Percentage of CD34⁺ (P < .001) and total (P < .001) hematogones in correlation with donor age. Time of neutrophil engraftment was significantly longer (P = .039) when total infused CD34⁺ cell content was <3 × 106/kg. CONCLUSIONS: A remarkable population of hematogones within the CD34⁺ cell pool was detected in bone marrow grafts. Detection of the ratio of hematogones and most primitive stem cells (CD34⁺CD90⁺CD38⁻) may overall provide more information to build a better correlation between CD34⁺ cell content and the recovery of bone marrow.

Significance of inhibitory maternal killer-cell immunoglobulin-like receptor (KIR) and fetal KIR ligand genotype combinations in placenta related obstetric complications.

Some maternal killer-cell immunoglobulin-like receptor (KIR) and fetal KIR ligand genotypes are associated with obstetric complications, such as recurrent miscarriage, fetal growth restriction, preeclampsia, and preterm birth. However, how KIR/KIR ligand genotypes affect these placenta-related obstetric complications has not been fully understood. We aimed to demonstrate the association of maternal KIR-fetal KIR ligand genotype combinations with immunological/metabolic risk factor associated placenta-related obstetric complications. This study consisted of three groups of pregnant women: 1) Miscarriage group (n = 30), 2) Complicated Pregnancy (CP) group (n = 30), and 3) Control group (n = 30). The observed maternal genotype frequencies of all inhibitory and activating KIRs were similar in all groups (p > 0.05). However, inhibitory 2DL3 was quite frequent in the miscarriage group (p = 0.052). There was no difference between groups in terms of centromeric and telomeric maternal haplotypes (p > 0.05). The fetal group 1 HLA-C genotype was frequently detected in the miscarriage and CP groups with rates of 83.3 % and 93.3 % respectively, while the observed frequency was 70 % in the control group. The fetal group 2 HLA-C genotype was the same in all groups. The results demonstrated significantly less fetal group 2 HLA-C homozygosity in the CP groups when compared to the control group (p = 0.020). The fetal HLA-Bw4 genotype was detected more frequently in the miscarriage and CP groups (p = 0.028 and p = 0.001, respectively). The inhibitory KIR/KIR ligand genotype combinations of 2DL3-C1 and 3DL1-Bw4 were more frequent in the miscarriage and CP groups (p = 0.045 and p = 0.002, respectively). Enhanced NK cell inhibition may be one of the mechanisms underlying placenta-related obstetric complications.

Toward In Vitro Epigenetic Drug Design for Thyroid Cancer: The Promise of PF-03814735, an Aurora Kinase Inhibitor.

Thyroid cancer (TC) is a very common malignancy worldwide. Chief among the innovative molecular drug targets for TC are epigenetic modifications. Increased telomerase activity in cancer cells makes telomerase a novel target for epigenetic anticancer drug innovation. Recently, telomerase reverse transcriptase (TERT) gene promoter (TERTp) mutations (C228T and C250T) were reported at high frequency in TC cell lines and tumor biopsies. In this study, three representative TC cell lines, mutant TERTp (TPC1), mutant BRAF/TERTp (KTC2), and wild-type TERTp (WRO), were screened with a drug library composed of 51 epigenetic drugs: 14 Aurora kinase inhibitors; 23 histone deacetylase inhibitors; 5 sirtuin modifiers; 3 hypoxia-inducible factor inhibitors; 2 DNA methyltransferase inhibitors; 2 histone methyltransferase inhibitors, a histone demethylase inhibitor, and a bromodomain inhibitor. Effects of the drugs on cell growth at 48 and 72 h were compared. PF-03814735, a small-molecule inhibitor of Aurora kinase A (IC50 = 0.8 nM) and B (IC50 = 5 nM), was the most potent on KTC2 cells, whereas CUDC-101, a multitarget inhibitor, was effective on both WRO and KTC2 cells. Notably, PF-03814735 was found to be the most effective epigenetic drug on cell lines harboring the C228T mutation. In conclusion, these new findings offer specific guidance on dose and time course selection to design novel therapeutic interventions against TC using PF-03814735, and specifically target cells carrying the TERTpC228T mutation. In a larger context of drug discovery science, these findings inform new strategies to forecast optimal treatment regimens for TC, particularly with Aurora kinase inhibitors and in ways guided by epigenetic drug design.

Investigating and targeting chronic lymphocytic leukemia metabolism with the human immunodeficiency virus protease inhibitor ritonavir and metformin.

Chronic lymphocytic leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity. Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal. Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with human immunodeficiency (HIV) protease inhibitor ritonavir, which inhibits GLUT4, elicits toxicity similar to that elicited upon glucose deprivation. CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for the treatment of diabetes in patients with HIV, demonstrating the tolerance to this combination in humans. Our studies strongly substantiate further investigation of Food and Drug Administration approved ritonavir and metformin for CLL.

Targeting the metabolic plasticity of multiple myeloma with FDA-approved ritonavir and metformin.

PURPOSE: We have previously demonstrated that ritonavir targeting of glycolysis is growth inhibitory and cytotoxic in a subset of multiple myeloma cells. In this study, our objective was to investigate the metabolic basis of resistance to ritonavir and to determine the utility of cotreatment with the mitochondrial complex I inhibitor metformin to target compensatory metabolism. EXPERIMENTAL DESIGN: We determined combination indices for ritonavir and metformin, impact on myeloma cell lines, patient samples, and myeloma xenograft growth. Additional evaluation in breast, melanoma, and ovarian cancer cell lines was also performed. Signaling connected to suppression of the prosurvival BCL-2 family member MCL-1 was evaluated in multiple myeloma cell lines and tumor lysates. Reliance on oxidative metabolism was determined by evaluation of oxygen consumption, and dependence on glutamine was assessed by estimation of viability upon metabolite withdrawal in the context of specific metabolic perturbations. RESULTS: Ritonavir-treated multiple myeloma cells exhibited increased reliance on glutamine metabolism. Ritonavir sensitized multiple myeloma cells to metformin, effectively eliciting cytotoxicity both in vitro and in an in vivo xenograft model of multiple myeloma and in breast, ovarian, and melanoma cancer cell lines. Ritonavir and metformin effectively suppressed AKT and mTORC1 phosphorylation and prosurvival BCL-2 family member MCL-1 expression in multiple myeloma cell lines in vitro and in vivo. CONCLUSIONS: FDA-approved ritonavir and metformin effectively target multiple myeloma cell metabolism to elicit cytotoxicity in multiple myeloma. Our studies warrant further investigation into repurposing ritonavir and metformin to target the metabolic plasticity of myeloma to more broadly target myeloma heterogeneity and prevent the reemergence of chemoresistant aggressive multiple myeloma.

Expression and phosphorylation of the AS160_v2 splice variant supports GLUT4 activation and the Warburg effect in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a fatal plasma cell malignancy exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e., the Warburg effect). We have previously demonstrated that myeloma cells exhibit constitutive plasma membrane (PM) localization of GLUT4, consistent with the dependence of MM cells on this transporter for maintenance of glucose consumption rates, proliferative capacity, and viability. The purpose of this study was to investigate the molecular basis of constitutive GLUT4 plasma membrane localization in MM cells. FINDINGS: We have elucidated a novel mechanism through which myeloma cells achieve constitutive GLUT4 activation involving elevated expression of the Rab-GTPase activating protein AS160_v2 splice variant to promote the Warburg effect. AS160_v2-positive MM cell lines display constitutive Thr642 phosphorylation, known to be required for inactivation of AS160 Rab-GAP activity. Importantly, we show that enforced expression of AS160_v2 is required for GLUT4 PM translocation and activation in these select MM lines. Furthermore, we demonstrate that ectopic expression of a full-length, phospho-deficient AS160 mutant is sufficient to impair constitutive GLUT4 cell surface residence, which is characteristic of MM cells. CONCLUSIONS: This is the first study to tie AS160 de-regulation to increased glucose consumption rates and the Warburg effect in cancer. Future studies investigating connections between the insulin/IGF-1/AS160_v2/GLUT4 axis and FDG-PET positivity in myeloma patients are warranted and could provide rationale for therapeutically targeting this pathway in MM patients with advanced disease.