c-MYB is a transcriptional regulator of ESPL1/Separase in BCR-ABL-positive chronic myeloid leukemia

Background: Genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML). Recently, we have shown that clonal evolution and blast crisis correlate with altered expression and activity of Separase, a cysteine endopeptidase that is a mitotic key player in chromosomal segregation and centriole duplication. Hyperactivation of Separase in human hematopoietic cells has been linked to a feedback mechanism that posttranslationally stimulates Separase proteolytic activity after imatinib therapy-induced reduction of Separase protein levels. Methods and Results: In search for potential therapy-responsive transcriptional mechanisms we have investigated the role of the transcription factor c-MYB for Separase expression in CML cell lines (LAMA-84, K562, BV-173) and in clinical samples. Quantitative RT-PCR and Western blot immunostaining experiments revealed that c-MYB expression levels are decreased in an imatinib-dependent manner and positively correlate with Separase expression levels in cell lines and in clinical CML samples. RNA silencing of c-MYB expression in CML cell lines resulted in reduced Separase protein levels. Gelshift and ChIP assays confirmed that c-MYB binds to a putative c-MYB binding sequence located within the ESPL1 promoter. Conclusions: Our data suggest that ESPL1/Separase is a regulatory target of c-MYB. Therefore, c-MYB, known to be required for BCR-ABL-dependent transformation of hematopoietic progenitors and leukemogenesis, may also control the Separase-dependent fidelity of mitotic chromosomal segregation and centriole duplication essential for maintenance of genomic stability

Dynamics of cytogenetic aberrations in Philadelphia chromosome positive and negative hematopoiesis during dasatinib therapy of chronic myeloid leukemia patients after imatinib failure

Clonal cytogenetic aberrations of the Philadelphia chromosome (Ph) positive hematopoiesis have been associated with the natural evolution of chronic myeloid leukemia (CML) to advanced disease. Clonal aberrations of Ph negative metaphases have been described after treatment with interferon or imatinib. This study evaluates the effect of dasatinib on Ph positive clones with additional cytogenetic aberrations and the frequency of novel aberrations in Ph positive and negative metaphases. Seventy-one patients treated with dasatinib after imatinib failure for a median of nine months were evaluated. Novel aberrations within Ph positive and negative clones appeared in six and three patients, respectively

Effect of ABCG2, OCT1, and ABCB1(MDR1) Gene Expression on Treatment-Free Remission in a EURO-SKI Subtrial

Introduction Tyrosine kinase inhibitors (TKIs) can safely be discontinued in chronic myeloid leukemia (CML) patients with sustained deep molecular response. ABCG2 (breast cancer resistance protein), OCT1 (organic cation transporter 1), and ABCB1 (multidrug resistance protein 1) gene products are known to play a crucial role in acquired pharmacogenetic TKI resistance. Their influence on treatment-free remission (TFR) has not yet been investigated. Materials and Methods RNA was isolated on the last day of TKI intake from peripheral blood leukocytes of 132 chronic phase CML patients who discontinued TKI treatment within the European Stop Tyrosine Kinase Inhibitor Study trial. Plasmid standards were designed including subgenic inserts of OCT1, ABCG2, and ABCB1 together with GUSB as reference gene. For expression analyses, quantitative real-time polymerase chain reaction was used. Multiple Cox regression analysis was performed. In addition, gene expression cutoffs for patient risk stratification were investigated. Results The TFR rate of 132 patients, 12 months after TKI discontinuation, was 54% (95% confidence interval [CI], 46%-62%). ABCG2 expression (‰) was retained as the only significant variable (P = .02; hazard ratio, 1.04; 95% CI, 1.01-1.07) in multiple Cox regression analysis. Only for the ABCG2 efflux transporter, a significant cutoff was found (P = .04). Patients with an ABCG2/GUSB transcript level >4.5‰ (n = 93) showed a 12-month TFR rate of 47% (95% CI, 37%-57%), whereas patients with low ABCG2 expression (≤4.5‰; n = 39) had a 12-month TFR rate of 72% (95% CI, 55%-82%). Conclusion In this study, we investigated the effect of pharmacogenetics in the context of a CML treatment discontinuation trial. The transcript levels of the efflux transporter ABCG2 predicted TFR after TKI discontinuation

Inhibition of lysyl oxidases synergizes with 5-azacytidine to restore erythropoiesis in myelodysplastic and myeloid malignancies

Limited response rates and frequent relapses during standard of care with hypomethylating agents in myelodysplastic neoplasms (MN) require urgent improvement of this treatment indication. Here, by combining 5-azacytidine (5-AZA) with the pan-lysyl oxidase inhibitor PXS-5505, we demonstrate superior restoration of erythroid differentiation in hematopoietic stem and progenitor cells (HSPCs) of MN patients in 20/31 cases (65%) versus 9/31 cases (29%) treated with 5-AZA alone. This effect requires direct contact of HSPCs with bone marrow stroma components and is dependent on integrin signaling. We further confirm these results in vivo using a bone marrow niche-dependent MN xenograft model in female NSG mice, in which we additionally demonstrate an enforced reduction of dominant clones as well as significant attenuation of disease expansion and normalization of spleen sizes. Overall, these results lay out a strong pre-clinical rationale for efficacy of combination treatment of 5-AZA with PXS-5505 especially for anemic MN

The Chromosomal Basis of Cancer

Conventional genetic theories have failed to explain why cancer (1) is not heritable and thus extremely rare in newborns, (2) is caused by non-mutagenic carcinogens, (3) develops only years to decades after initiation by carcinogens, (4) follows pre-neoplastic aneuploidy, (5) is aneuploid, (6) is chromosomally and phenotypically “unstable”, (7) carries specific aneusomies, (8) generates much more complex phenotypes than conventional mutation such as multidrug resistance, (9) generates nonselective phenotypes such as metastasis (no benefit at native site) and “immortality” (not necessary for tumorigenesis), and (10) does not contain carcinogenic mutations. We propose, instead, that cancer is a chromosomal disease. Accordingly carcinogenesis is initiated by random aneuploidies, which are induced by carcinogens or spontaneously. Since aneuploidy unbalances 1000s of genes, it corrupts teams of proteins that segregate, synthesize and repair chromosomes. Aneuploidy is therefore a steady source of chromosomal variations from which, in classical Darwinian terms, selection encourages the evolution and malignant progression of cancer cells. The rates of specific chromosomal variations can exceed conventional mutations by 4–11 orders of magnitude, depending on the degrees of aneuploidy. Based on their chromosomal constitution cancer cells are new cell “species” with specific aneusomies, but unstable karyotypes. The cancer-specific aneusomies generate complex, malignant phenotypes through the abnormal dosages of 1000s of genes, just as trisomy 21 generates Down syndrome. In sum, cancer is caused by chromosomal disorganization, which increases karyotypic entropy. Thus, cancer is a chromosomal rather than a genetic disease. The chromosomal theory explains (1) non-heritable cancer because aneuploidy is not heritable, (2) non-mutagenic carcinogens as aneuploidogens, (3) long neoplastic latencies by the low probability of evolving new species, (4) nonselective phenotypes via genes hitchhiking with selective chromosomes, and (5) immortality because, through their cellular heterogeneity, cancers survive negative mutations and cytotoxic drugs via resistant subspecies