Monospecific antibody directed against bovine brain tubulin was used as an immunofluorescent probe to evaluate the distribution of microtubules in normal and transformed cells grown in tissue culture. The fluorescent staining pattern of transformed and nontransformed cells is significantly different and may be used in conjunction with other morphological features to identify transformants in mixed cell populations. Normal cells are flattened, elongated, and fibroblastic; they display numerous Colcemid-sensitive fluorescent cytoplasmic filaments, presumably microtubules. Transformed cells, however, are smaller, more polygonal in shape, and contain very few cytoplasmic tubules. During mistosis the cytoplasmic microtubule complex of normal cells completely disappears, but reappears after cell division. Treatment of transformed cells with dibutyry-adenosine 3':5'-cyclic monophosphate plus testosterone or theophylline restores the normal fibroblastic appearance of the cells and stimulates the assembly of numerous cytoplasmic microtubules. This study provides further evidence for two separate microtubule entities in cycling nontransformed cells: a cytoplasmic microtubule complex and the microtubules of the mitotic spindle. Although an interchange of tubulin dimers seems to exist between microtubules in the two systems, control of tubule assembly may be under separate constraints. Stimulation of cytoplasmic microtuble assembly in transformed cells by derivatives of adenosine 3':5'-cycle monophosphate suggests that impairment of the cytoplasmic microtubule complex in these cells may be due to suboptimal levels of adenosine 3':5'-cyclic monophosphate.

Hox homeobox transcription factors drive leukemogenesis efficiently only in the presence of Meis or Pbx proteins. Here we show that Pbx3 and Meis1 need to dimerize to support Hox-induced leukemia and we analyze the molecular details of this cooperation. In the absence of Pbx3, Meis1 was highly unstable. As shown by a deletion analysis Meis1 degradation was contingent on a motif coinciding with the Pbx-binding domain. Either deletion of this sequence or binding to Pbx3 prolonged the half-life of Meis1 by preventing its ubiquitination. Meis1 break-down could also be blocked by inhibition of the ubiquitin proteasome system, indicating tight post-transcriptional control. In addition, Meis1 and Pbx3 cooperated genetically as overexpression of Pbx3 induced endogenous Meis1 transcription. These functional interactions translated into in vivo activity. Blocking Meis1/Pbx3 dimerization abrogated the ability to enhance proliferation and colony-forming cell numbers in primary cells transformed by Hoxa9. Furthermore, expression of Meis1 target genes Flt3 and Trib2 was dependent on Pbx3/Meis1 dimerization. This correlated with the requirement of Meis1 to bind Pbx3 in order to form high affinity DNA/Hoxa9/Meis1/Pbx3 complexes in vitro. Finally, kinetics and severity of disease in transplantation assays indicated that Pbx3/Meis1 dimers are rate-limiting factors for Hoxa9-induced leukemia. Pbx3 and Meis1 cooperate through multiple mechanisms to support Hox-induced murine leukemiaMaria-Paz Garcia-Cuellar, Julia Steger, Elisa Füller, Katrin Hetzner, and Robert K. Slany Department of Genetics, Friedrich-Alexander-University, Erlangen, Germany ABSTRACT ments the ability of Meis1 to support Hox-mediated leukemogenesis. We demonstrate that Pbx3 protects Meis1 from proteasomal degradation. Additionally, Pbx3 increases Meis1 affinity for Hoxa9 and it induces endogenous Meis1 transcription. MethodsPlasmids, retroviral constructs, antibodies and cell culture

Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia.

HOX homeobox proteins are key oncogenic drivers in hematopoietic malignancies. Here we demonstrate that HOXA1, HOXA6 and predominantly HOXA9 are able to induce the production of insulin-like growth factor 1 (Igf1). In chromatin immunoprecipitations, HOXA9 bound directly to the putative promoter and a DNase-hypersensitive region in the first intron of the Igf1 gene. Transcription rates of the Igf1 gene paralleled HOXA9 activity. Primary cells transformed by HOXA9 expressed functional Igf1 receptors and activated the protein kinase Akt in response to Igf1 stimulation, suggesting the existence of an autocrine signaling loop. Genomic deletion of the Igf1 gene by Cre-mediated recombination increased sensitivity toward apoptosis after serum starvation. In addition, the leukemogenic potential of Igf1-negative, HOXA9-transformed cells was impaired, leading to a significant delay in disease development on transplantation into recipient animals.

Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations that frequently occur in utero in preleukemic cells. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell ALL. A previous study suggested that ETV6-RUNX1 positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1 positive ALL in children by a factor of 100. This finding indicated that secondary, but relatively rare cooperating oncogenic hits are necessary for the development of overt leukemia. However, later studies could not confirm this high incidence of preleukemic cells in newborns. To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based screening of ≈1000 cord blood samples from healthy newborns. The GIPFEL method is capable to detect the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In the case of ETV6-RUNX1 positive leukemia, GIPFEL exploits the unique presence of a genomic fragment joining material from chromosomes 12 and 21 in the translocation-positive cells. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. Importantly, the ligation site is independent of the translocation point within the individual DNA circle. The published breakpoint regions of the ETV6 and RUNX1 genes involved in the translocation were analyzed in silico for restriction sites that allow digestion of all possible translocation events to yield fragments smaller than approximately 50 kb. This condition was met for ETV6-RUNX1 breakpoints by digestion with SacI. Primer pairs were designed amplifying the complete set of theoretically predicted circularized fragments requiring 36 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from cell lines, diagnostic specimens from ALL patients, peripheral blood from healthy donors and cord blood samples from newborns by column purification. The equivalent of approximately 4x105 cells (2.5 µg DNA) was subjected to the SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In a first proof-of-principle study employing the ETV6-RUNX1 translocation positive cell line REH, process optimization close to the theoretical limits was carried out. Cell dilution and mixing studies revealed that under optimal conditions approximately 40 translocation positive cells (=10-4) present in the input DNA are sufficient to produce a reliable output signal. The method was next tested in a blinded study with 60 samples obtained from ETV6-RUNX1 diagnostic ALL samples. ETV6-RUNX1 samples positive at 10-4, being diluted from these diagnostic samples, still gave a reliable output signal. There was no false positive result. Detection coverage (=sensitivity) was 64%. This method was then applied to a retrospective sample set of cryopreserved anonymized cord blood samples of ≈1000 healthy newborns to determine frequency and levels of translocation-positive cells. First results will be presented. In conclusion this population-based study will allow an estimate of the actual incidence of ETV6-RUNX1 positive preleukemic cells in healthy newborns. The results will enable us to evaluate the penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and will provide a basis for the assessment of potential secondary environmental or spontaneously occurring cooperating oncogenic lesions in ETV6-RUNX1 positive childhood leukemia. Disclosures No relevant conflicts of interest to declare.