Nathalie Sorel

Leukemic stem cell persistence in chronic myeloid leukemia patients with sustained undetectable molecular residual disease.

Sustained undetectable molecular residual disease (UMRD) is obtained in a minority of patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors. It remains unclear whether these patients are definitively cured of their leukemia or whether leukemic stem cells (LSCs) persist in their BM. We have evaluated the presence of BCR-ABL-expressing marrow LSCs in 6 patients with chronic myeloid leukemia with sustained UMRD induced by IFN-α (n = 3), imatinib mesylate after IFN-α failure (n = 2), and dasatinib after imatinib intolerance (n = 1). Purified CD34(+) cells were used for clonogenic and long-term culture-initiating cell assays performed on classic or HOXB4-expressing MS-5 feeders. Using this strategy, we identified BCR-ABL-expressing LSCs in all patients. Interestingly, long-term culture-initiating cell assays with MS-5/HOXB4 stromal feeders increased detected numbers of LSCs in 3 patients. The relation between LSC persistency and a potential risk of disease relapse for patients with durable UMRD (on or off tyrosine kinase inhibitor therapy) warrants further investigation.

Neutrophil gelatinase-associated lipocalin expression in chronic myeloid leukemia.

The murine equivalent of neutrophil gelatinase-associated lipocalin (NGAL) was previously found to be increased by BCR-ABL expression in murine models of chronic myeloid leukemia (CML). Our study evaluates, in CML patients at various clinical stages, the levels of NGAL mRNA in blood samples and protein in sera. A highly significant increase of mRNA expression and protein secretion was shown in patients at diagnosis. The parallel expression of NGAL and BCR-ABL at the early stage of CML process allows us to suggest that NGAL could play an important role in the physiopathology of CML.

Quantitative monitoring of the T315I mutation in patients with chronic myeloid leukemia (CML)

Tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment of chronic myeloid leukemia (CML). However, resistances are occasionally observed, mainly due to mutations within the BCR-ABL kinase domain. The T315I substitution confers complete resistance to TKIs commonly used in clinical practice. In the present study, we used an allele-specific quantitative-RT-PCR to perform a molecular follow-up of BCR-ABL transcripts harboring the T315I mutation. We retrospectively quantified BCR-ABL315I mRNA in five patients who acquired the T315I mutation. Our results highlight the relevance of allele-specific Q-RT-PCR experiments for the monitoring of mutated BCR-ABL transcripts and suggest that the kinetics of emergence of T315I mutant mRNA is influenced by the stage of the disease and the presence of previous BCR-ABL kinase domain mutations.

BCR-ABL expression in leukemic progenitors and primitive stem cells of patients with chronic myeloid leukemia

To the editor:nnKumari and colleagues have recently reported a detailed analysis of BCR-ABL expression in CFU-Cs (colony forming units in culture) of patients with chronic myeloid leukemia (CML).[1][1] Using quantitative reverse-transcription PCR on individual Ph1 hematopoietic colonies, they

The downregulation of BAP1 expression by BCR-ABL reduces the stability of BRCA1 in chronic myeloid leukemia.

BCR-ABL induces an intrinsic genetic instability in chronic myeloid leukemia (CML). The protein breast cancer 1, early onset (BRCA1)-associated protein 1 (BAP1) is a deubiquitinase interacting with the DNA repair regulator BRCA1 and is frequently inactivated in many cancers. Here, we report that BAP1 mRNA and protein levels are downregulated in a BCR-ABL1-expressing hematopoietic cell line (UT-7/11). A decrease of BAP1 transcripts is also observed in newly diagnosed CML patients. Moreover, BAP1 protein levels are low or undetectable in CD34(+) cells from CML patients at diagnosis as compared with CD34(+) cells from normal donors. In addition, BRCA1 protein level is reduced in BCR-ABL1-expressing UT-7/11 cells. Finally, the enforced expression of BAP1 is associated with BRCA1 protein deubiquitination and restoration. These results demonstrate BAP1 as a major link with the BCR-ABL-induced downregulation of BRCA1 in CML.

Biological effects of T315I-mutated BCR-ABL in an embryonic stem cell–derived hematopoiesis model

The occurrence of T315I mutation during the course of targeted therapies of chronic myeloid leukemia is a major concern because it confers resistance to all currently approved tyrosine kinase inhibitors. The exact phenotype of the hematopoietic stem cell and the hierarchical level of the occurrence of this mutation in leukemic hematopoiesis has not been determined. To study the effects of T315I-mutated breakpoint cluster region-abelson (BCR-ABL) in a primitive hematopoietic stem cell, we have used the murine embryonic stem cell (mESC)-derived hematopoiesis model. Native and T315I-mutated BCR-ABL were introduced retrovirally in mESC-derived embryonic bodies followed by induction of hematopoiesis. In several experiments, T315I-mutated and nonmutated BCR-ABL-transduced embryonic bodies rapidly generated hematopoietic cells on OP-9 feeders, with evidence of hematopoietic stem cell markers. After injection into NOD/SCID mice, these cells induced myeloid and lymphoid leukemias, whereas transplantation of control (nontransduced) hematopoietic cells failed to produce any hematopoietic reconstitution inxa0vivo. Moreover, the expression of native and T315I-mutated BCR-ABL conferred to mESC-derived hematopoietic cells a self-renewal capacity demonstrated by the generation of leukemias after secondary transplantations. Secondary leukemias were more aggressive with evidence of extramedullary tumors. The expression of stem cell regulator Musashi-2 was found to be increased in bone marrow of leukemic mice. These data show that T315I-mutated BCR-ABL is functional at the stem cell level, conferring to mESC-derived leukemic cells a long-term hematopoietic repopulation ability. This model could be of interest to test the efficiency of drugs at the stem cell level in leukemias with T315I mutation.

Superoxide dismutase 2 (SOD2) contributes to genetic stability of native and T315I-mutated BCR-ABL expressing leukemic cells

Manganese Superoxide dismutase 2 (SOD2) plays a crucial role in antioxidant defense but there are no data suggesting its role in genetic instability in CML. We evaluated the effects of SOD2 silencing in human UT7 cell line expressing either non-mutated or T315I-mutated BCR-ABL. Array-CGH experiments detected in BCR-ABL-expressing cells silenced for SOD2 a major genetic instability within several chromosomal loci, especially in regions carrying the glypican family (duplicated) and β-defensin genes (deleted). In a large cohort of patients with chronic myeloid leukemia (CML), a significant decrease of SOD2 mRNA was observed. This reduction appeared inversely correlated with leukocytosis and Sokal score, high-risk patients showing lower SOD2 levels. The analysis of anti-oxidant gene expression analysis revealed a specific down-regulation of the expression of PRDX2 in UT7-BCR-ABL and UT7-T315I cells silenced for SOD2 expression. Gene set enrichment analysis performed between the two SOD2-dependent classes of CML patients revealed a significant enrichment of Reactive Oxygen Species (ROS) Pathway. Our data provide the first evidence for a link between SOD2 expression and genetic instability in CML. Consequently, SOD2 mRNA levels should be analyzed in prospective studies as patients with low SOD2 expression could be more prone to develop a mutator phenotype under TKI therapies.

ABL-Kinase Mutations in Progenitors and Stem Cells from Chronic Myeloid Leukemia Patients

Tyrosine kinase inhibitors (TKIs) have profoundly changed the natural history and prognosis of chronic myeloid leukemia (CML). However, a small proportion of patients develop a resistance towards targeted therapies. Missense mutations located within the kinase domain of the BCR-ABL oncogene (also referred to as BCR-ABL mutations or ABL-kinase mutations) are the most common mechanism of resistance. Although it has clearly been established that genetic instability inherent to BCR-ABL expressing leukemic cells predisposes the latter to the acquisition of mutations, the hierarchical distribution of these mutations in stem cells was not known until recently. There is now evidence suggesting that ABL-kinase mutations occur in hematopoietic progenitors and stem cells expressing the BCR-ABL oncogene, adding therefore an increased level of complexity to the phenomenon of TKI-resistance in CML stem cells.