Intrabone transplant provides full stemness of cord blood stem cells with fast hematopoietic recovery and low GVHD rate: results from a prospective study

Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC > 0.5 × 109/L) and 70 ± 10% (platelet > 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ±  9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche

In vitro and in vivo single-agent efficacy of checkpoint kinase inhibition in acute lymphoblastic leukemia

Background: Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Methods: The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B-/T-ALL cell lines. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent n-ethyl-n-nitrosourea. Results: Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for \u3b3-H2A.X (Ser139) in 68 % of ALL patients. In human B-and T-ALL cell lines, inhibition of Chk1/2 as a single treatment strategy efficiently triggered the Chk1-Cdc25-Cdc2 pathway resulting in a dose-and time-dependent cytotoxicity, induction of apoptosis, and increased DNA damage. Moreover, treatment with PF-00477736 showed efficacy ex vivo in primary leukemic blasts separated from 14 adult ALL patients and in vivo in mice transplanted with T-ALL, arguing in favor of its future clinical evaluation in leukemia. Conclusions: In vitro, ex vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation

Novel and Rare Fusion Transcripts Involving Transcription Factors and Tumor Suppressor Genes in Acute Myeloid Leukemia.

Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line

The human Smoothened inhibitor PF-04449913 induces exit from quiescence and loss of multipotent Drosophila hematopoietic progenitor cells

International audienceThe efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells and the prevention of disease relapse through targeting pathways that stimulate generation and maintenance of these cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In Drosophila melanogaster the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. Using Drosophila as an in vivo model, we investigated the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. We conclude that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether our results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies

A case report of acute myeloid leukemia and neurofibromatosis 1

We report a case of a 65-year old patient affected by neurofibromatosis 1, documented by the presence of germ-line mutation on the <em>NF1</em> gene, who developed various hyperproliferative malignant and benign diseases. He was brought to our attention for the diagnosis of acute myeloid leukemia revealed by major fatigue and dyspnea. The disease characteristics at diagnosis were hyperleukocytosis and complex karyotype with the inversion of the chromosome 16, classifying as a high-risk leukemia. The association between leukemia and neurofibromatosis 1 is controversial and needs to be further investigated. Nevertheless, such patients present a wide number of comorbidities that make therapeutic strategies most difficult

BCR-ABL1 transcript detection in patients with Philadelphia-positive Acute Lymphoblastic Leukemia (ALL) using a new and high sensitive nanofluidic method

Background: Detection of residual leukemic cells by measuring BCR-ABL1 transcript level with assays based on quantitative polymerase chain reaction (qPCR) is necessary in the monitoring of minimal residual disease in patients with BCR-ABL1 positive ALL. Aim: To investigate the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies, we compared results obtained by conventional qPCR with those obtained by the nanofluidic approach. Patients and methods: We analyzed 60 BCR-ABL1 positive ALL samples expressing p190 (42) or p210 (18) isoform, first by TaqMan absolute qPCR (Applied Biosystems 7900HT Fast Real-Time PCR System). BCR-ABL1 target gene and ABL1 control gene copies were derived by the interpolation of cycle threshold values to the appropriate standard curve obtained using serial plasmid dilutions containing known BCR-ABL1 or ABL1 gene copies. Results: Samples resulted in complete (22) or major (38) molecular response (BCR-ABL1/ABL1 ratio 6a 0.001 or < 0.1, respectively; 3,8 BCR-ABL1 mean copy number copies). Subsequently we reanalyzed the samples using the 12.765 Digital array (Fluidigm) and the integrated BioMark Digital PCR Analysis software (Fluidigm). The nanofluidic biochip consists in twelve panels, each containing 765 individual reaction chambers where samples are portioned prior to qPCR. As fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Sensitivity and reproducibility of the assay were assessed using six serial dilutions of plasmids (Ipsogen) expressing known BCR-ABL1 p190 transcript copy number (10000; 1000; 100; 50; 10; 1 copies). A 2 \ub5l volume of input cDNA was loaded and two panel for each dilution were used. Results showed a detection rate until a copy of target sequence and a pairing significantly effective between replicates. We then analyzed duplicates of BCR-ABL1 ALL samples: digital array resulted positive in 21 of major molecular response samples (2,13 BCR-ABL1 mean copy number copies) and in any complete molecular response samples. In conclusion, the Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript, and it could provide an accurate monitoring method for BCR-ABL1-positive ALL CR patients. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione \u2013 Universit\ue0 2007 \u2013 2009

The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia

none11nononeAndrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Enrico Derenzini, Anna Ferrari, Valentina Robustelli, Viviana Guadagnuolo, Cristina Papayannidis, Maria Chiara Abbenante, Sandro Grilli, Giovanni MartinelliAndrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Enrico Derenzini, Anna Ferrari, Valentina Robustelli, Viviana Guadagnuolo, Cristina Papayannidis, Maria Chiara Abbenante, Sandro Grilli, Giovanni Martinell