Tyrosine kinase inhibitors (TKI) have become a first‐line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34⁺lin⁻) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF‐κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G₀ and G₂ phases. Furthermore, we found cell cycle inhibition to correlate with down‐regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF‐κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.

Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease. In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34⁺CD38⁻lin⁻ LSC and HSC. Our results demonstrate that cellular location of p18^INK4c and p57^Kip2 seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18^INK4c and p57^Kip2 nuclear location. The differences in p18^INK4cand p57^Kip2activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.

Ph-like subtypes with CRLF2 abnormalities are frequent among Hispano–Latino children with pre-B ALL. Therefore, there is solid ground to suggest that this subtype is frequent in Mexican patients. The genomic complexity of Ph-like subtype constitutes a challenge for diagnosis, as it requires diverse genomic methodologies that are not widely available in diagnostic centers in Mexico. Here, we propose a diagnostic strategy for Ph-like ALL in accordance with our local capacity. Pre-B ALL patients without recurrent gene fusions (104) were classified using a gene-expression profile based on Ph-like signature genes analyzed by qRT-PCR. The expressions of the CRLF2 transcript and protein were determined by qRT-PCR and flow cytometry. The P2RY8::CRLF2, IGH::CRLF2, ABL1/2 rearrangements, and Ik6 isoform were screened using RT-PCR and FISH. Surrogate markers of Jak2-Stat5/Abl/Ras pathways were analyzed by phosphoflow. Mutations in relevant kinases/transcription factors genes in Ph-like were assessed by target-specific NGS. A total of 40 patients (38.5%) were classified as Ph-like; of these, 36 had abnormalities associated with Jak2-Stat5 and 4 had Abl. The rearrangements IGH::CRLF2,P2RY8::CRLF2, and iAMP21 were particularly frequent. We propose a strategy for the detection of Ph-like patients, by analyzing the overexpression/genetic lesions of CRLF2, the Abl phosphorylation of surrogate markers confirmed by gene rearrangements, and Sanger sequencing.

In this study, we determined the gene expression profiles of bone marrow‐derived cell fractions, obtained from normal subjects and Chronic Myeloid Leukemia (CML) patients, that were highly enriched for hematopoietic stem (HSCs) and progenitor (HPCs) cells. Our results indicate that the profiles of CML HSCs and HPCs were closer to that of normal progenitors, whereas normal HSCs showed the most different expression profile of all. We found that the expression profiles of HSCs and HPCs from CML marrow were closer to each other than those of HSCs and HPCs from normal marrow. The major biologic processes dysregulated in CML cells included DNA repair, cell cycle, chromosome condensation, cell adhesion, and the immune response. We also determined the genomic changes in both normal and CML progenitor cells under culture conditions, and found that several genes involved in cell cycle, steroid biosynthesis, and chromosome segregation were upregulated, whereas genes involved in transcription regulation and apoptosis were downregulated. Interestingly, these changes were the same, regardless of the addition of Imatinib (IM) to the culture. Finally, we identified three genes—PIEZO2, RXFP1, and MAMDC2‐ that are preferentially expressed by CML primitive cells and that encode for cell membrane proteins; thus, they could be used as biomarkers for CML stem cells.

Venous thromboembolic disease (VTD) is a public health problem. We recently reported that endothelial colony-forming cells (ECFCs) derived from endothelial cells (EC) (ECFC-ECs) from patients with VTD have a dysfunctional state. For this study, we proposed that a dysfunctional status of these cells generates a reduction of its proliferative ability, which is also associated with senescence and reactive oxygen species (ROS). Human mononuclear cells (MNCs) were obtained from peripheral blood from 40 healthy human volunteers (controls) and 50 patients with VTD matched by age (20−50 years) and sex to obtain ECFCs. We assayed their proliferative ability with plasma of patients and controls and supernatants of cultures from ECFC-ECs, senescence-associated β-galactosidase (SA-β-gal), ROS, and expression of ephrin-B2/Eph-B4 receptor. Compared with cells from controls, cells from VTD patients showed an 8-fold increase of ECFCs that emerged 1 week earlier, reduced proliferation at long term (39%) and, in passages 4 and 10, a highly senescent rate (30±1.05% vs. 91.3±15.07%, respectively) with an increase of ROS and impaired expression of ephrin-B2/Eph-4 genes. Proliferation potential of cells from VTD patients was reduced in endothelial medium [1.4±0.22 doubling population (DP)], control plasma (1.18±0.31 DP), or plasma from VTD patients (1.65±0.27 DP). As compared with controls, ECFC-ECs from individuals with VTD have higher oxidative stress, proliferation stress, cellular senescence, and low proliferative potential. These findings suggest that patients with a history of VTD are ECFC-ECs dysfunctional that could be associated to permanent risk for new thrombotic events.