FLT3 mutations in canine acute lymphocytic leukemia

<p>Abstract</p> <p>Background</p> <p>FMS-like tyrosine kinase 3 (FLT3) is a commonly mutated protein in a variety of human acute leukemias. Mutations leading to constitutively active FLT3, including internal tandem duplications of the juxtamembrane domain (ITD), result in continuous cellular proliferation, resistance to apoptotic cell death, and a poorer prognosis. A better understanding of the molecular consequences of FLT3 activation would allow improved therapeutic strategies in these patients. Canine lymphoproliferative diseases, including lymphoma and acute leukemias, share evolutionarily conserved chromosomal aberrations and exhibit conserved mutations within key oncogenes when compared to their human counterparts. A small percentage of canine acute lymphocytic leukemias (ALL) also exhibit <it>FLT3 </it>ITD mutations.</p> <p>Methods</p> <p>We molecularly characterized <it>FLT3 </it>mutations in two dogs and one cell line, by DNA sequencing, gene expression analysis via quantitative real-time PCR, and sensitivity to the FLT3 inhibitor lestaurtinib via <it>in vitro </it>proliferation assays. FLT 3 and downstream mediators of FLT3 activation were assessed by Western blotting.</p> <p>Results</p> <p>The canine B-cell leukemia cell line, GL-1, and neoplastic cells from 2/7 dogs diagnosed cytologically with ALL were found to have <it>FLT3 </it>ITD mutations and <it>FLT3 </it>mRNA up-regulation. Lestaurtinib, a small molecule FLT3 inhibitor, significantly inhibited the growth of GL-1 cells, while not affecting the growth of two other canine lymphoid cell lines without the <it>FLT3 </it>mutation. Finally, western blots were used to confirm the conserved downstream mediators of <it>FLT3 </it>activating mutations.</p> <p>Conclusions</p> <p>These results show that ALL and FLT3 biology is conserved between canine and human patients, supporting the notion that canine ALL, in conjunction with the GL-1 cell line, will be useful in the development of a relevant large animal model to aid in the study of human FLT3 mutant leukemias.</p

Immunoprofiling of leukemic stem cells CD34+/CD38−/CD123+ delineate FLT3/ITD-positive clones

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous clonal disorder presenting with accumulation of proliferating undifferentiated blasts. Xenograft transplantation studies have demonstrated a rare population of leukemia-initiating cells called leukemic stem cells (LSCs) capable of propagating leukemia that are enriched in the CD34+/CD38− fraction. LSCs are quiescent, resistant to chemotherapy and likely responsible for relapse and therefore represent an ideal target for effective therapy. LSCs are reported to overexpress the alpha subunit of the IL-3 receptor (CD123) compared to normal CD34+/CD38− hematopoietic stem cells. It has not been demonstrated whether CD123-positive (CD34+/CD38−) subpopulation is enriched for any clonal markers of AML or any LSC properties. The aims of this study were to investigate whether FMS-like tyrosine kinase (FLT3)/internal tandem duplication (ITD) mutations are present at LSC level and whether FLT3/ITD mutation is confined to LSC as defined by CD34+/CD38−/CD123+ and not CD34+/CD38−/CD123−. Methods Thirty-four AML cases were analyzed by five-color flow cytometry and sequential gating strategy to characterize of CD34+/CD38−/CD123+ cells. These cells were sorted, analyzed by PCR, and sequenced for FLT3/ITD. Results In this study, we confirm significant expression of CD123 in 32/34 cases in the total blast population (median expression = 86 %). CD123 was also expressed in the CD34+/CD38− cells (96 ± 2 % positive) from 28/32 for CD123+ AML. CD123 was not expressed/low in normal bone marrow CD34+/CD38− cells (median expression = 0 %, range (0–.004 %). AML samples were tested for FLT3/ITD (10 positive/25). FLT3/ITD+ AML cases were sorted into two putative LSC populations according to the expression of CD123 and analyzed for FLT3/ITD again in the stem cell fractions CD34+/CD38−/CD123+ and CD34+/CD38−/CD123−. Interestingly, FLT3/ITD was only detected in CD34+/CD38−/CD123+ (7/7) and not in CD34+/CD38−/CD123− subpopulation (6/7). Conclusions This finding shows that FLT3/ITD are present at LSC level and may be a primary and not secondary event in leukemogenesis, and the oncogenic events of FLT3/ITD happen at a cell stage possessing CD123. It shows that CD123 immunoprofiling provides further delineation of FLT3+ LSC clone. This novel finding provides a rationale for treatment involving CD123-targeting antibodies with intracellular FLT3 inhibitors directed against CD34+/CD38−/CD123+. This may result in more effective anti-LSC eradication

Survey of Activated FLT3 Signaling in Leukemia

Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia

Locomotion disorders and skin and claw lesions in gestating sows housed in dynamic versus static groups

Lameness and lesions to the skin and claws of sows in group housing are commonly occurring indicators of reduced welfare. Typically, these problems are more common in group housing than in individual housing systems. Group management type (dynamic versus static) and stage of gestation influence the behavior of the animals, which in turn influences the occurrence of these problems. The present study compared prevalence, incidence and mean scores of lameness and skin and claw lesions in static versus dynamic group housed sows at different stages of gestation during three consecutive reproductive cycles. A total of 10 Belgian sow herds were monitored; 5 in which dynamic groups and 5 in which static groups were utilized. All sows were visually assessed for lameness and skin lesions three times per cycle and the claws of the hind limbs were assessed once per cycle. Lameness and claw lesions were assessed using visual analogue scales. Static groups, in comparison with dynamic groups, demonstrated lower lameness scores (P<0.05) and decreased skin lesion prevalence (24.9 vs. 47.3%, P<0.05) at the end of gestation. There was no difference between treatment group regarding claw lesion prevalence with 75.5% of sows demonstrating claw lesions regardless of group management. Prevalences of lameness (22.4 vs. 8.9%, P<0.05) and skin lesions (46.6 vs. 4.4%, P<0.05) were highest during the group-housed phase compared to the individually housed phases. Although the prevalence of lameness and skin lesions did not differ three days after grouping versus at the end of the group-housing phase, their incidence peaked during the first three days after moving from the insemination stalls to the group. In conclusion, the first three days after grouping was the most risky period for lameness incidence, but there was no significant difference between static or dynamic group management