The primary interests of Dr. Calabretta's laboratory are in the analysis of the role of the Myb gene family in the regulation of hematopoietic proliferation, differentiation, and survival and in the investigation of the molecular mechanisms underlying BCR/ABL-mediated leukemogenesis.  The proto-oncogene c-myb is a transcriptional factor specifically interacting with myb binding sites in the promoter region of target genes. Candidate target genes are those encoding the CD34 antigen and the c-kit receptor whose expression defines the hematopoietic stem cell.  Sequence analysis of the promoter of both genes revealed the presence of Myb binding sites; gel shift experiments demonstrated interaction with bacterially synthesized Myb protein; expression of c-myb in CD34-/c-kit- cells induced CD34 and c-kit expression. Other transcriptional factors of the ets family cooperate with c-myb in regulating CD34 and c-kit expression. 

The c-myb proto-oncogene is also involved in protecting hematopoietic cells from apoptosis induced by growth factor deprivation. c-myb dependent protection from apoptotic cell death involves upregulation of bcl-2 protein levels by a transcriptional mechanism. The Myb-responsive segment of the bcl-2 promoter has been identified and studies are now in progress to define the precise mechanism of promoter regulation. 

B-myb, a c-myb-related gene recently cloned in the laboratory, appears to function as a c-myb equivalent in non-hematopoietic cells. B-myb expression appears to be important in regulating the proliferation and survival of neuroblastoma cells. Recent studies show that B-myb protects IL-2 dependent T-lymphocytes from apoptosis induced by growth factor deprivation and by other death-inducing stimuli. Expression of Bcl-2 is enhanced by a transcription mechanism involving interaction of B-myb with a Myb binding site in the Bcl-2 promoter. Interestingly, the mechanism of Bcl-2 regulation is distinct from that involving c-myb. Transgenic mice in which Bcl-2 expression was directed to the T-cell compartment are now monitored to assess the in vivo consequences of ectopic B-myb expression. A-myb is another member of the Myb family homologous to c-myb in the DNA binding domain. Ectopic A-myb expression in transgenic mice induces lymphoma formation. Investigation of the mechanisms associated with tumor induction by A-myb is in progress.

Patients with chronic myelogenous leukemia carry the Philadelphia chromosome translocation characterized by generation of chimeric transcripts resulting from the juxtaposition of the c-abl and BCR genes. Dr. Calabretta's laboratory has shown that antisense oligodeoxynucleotides targeted to the breakpoint junction suppress in CML growth in vitro and in SCID mice. Dr. Calabretta's laboratory is presently assessing the role of potential downstream effectors of the Bcr/Abl tyrosine kinase that may be responsible for the phenotype of BCR/ABL-expressing cells. This phenotype includes enhanced survival and proliferation, differentiation arrest, and altered motility. We are studying several effectors implicated in these processes. For example, the PI-3k/Akt pathway is required for survival, while Rac-1 is required for the motility of BCR/ABL-expressing cells. BCR/ABL-dependent leukemogenesis is suppressed by expression of Akt and Rac dominant-negative mutants. Akt is involved in anti-apoptotic pathways by enhancing the activity of Raf-1 by a PKC-dependent mechanism and by inhibiting the pro-apoptotic BAD. BCR/ABL maintains BAD in its phosphorylated, inactive form not only by activation of BAD kinase(s), but also by suppressing the PP-1a serine phosphatase that dephosphorylates BAD. The identification of BCR/ABL-dependent pathways required for leukemogenesis is necessary for developing novel and more rational therapies for CML.

Keywords:  oncogenes, proliferation, apoptosis, gene-targeted therapy

PubMed Link For Calabretta B

Grassilli, E., Salomoni, P., Perrotti, D., Franceschi, C. and Calabretta, B. Resistance to apoptosis in CTLL-2 cells overexpressing B-Myb is associated with B-Myb-dependent bcl-2 induction. Cancer Res., 59:2451-2456, 1999.

Raschella, G., Cesi, V., Amendola, R., Negroni, A., Tanno, B., Altavista, P., Tonini, G.P., De Bernardi, B. and Calabretta, B. Expression of B-myb in neuroblastoma tumors in a poor prognostic factor independent from MYCN amplification. Cancer Res., 
59:3365-3368,1999.

Sevignani, C., Cranston, A., Iozzo, R.V., Fishel, R., and Calabretta, B. Spontaneous and mutagen-induced transformation of primary cultures of Msh2-/- p53-/- colonocytes. Cancer Res., 59:5882-5886, 1999.

Manzella, L., Gualdi, R., Perrotti, D., Nicolaides, N.C., Girlando, Giuseppe, G., Giuffrida, M.A., Messina, A., and Calabretta, B. The Interferon Regulatory Factors 1 and 2 Bind to a Segment of the Human c-myb First Intron: Possible Role in the Regulation of c-myb Expression. Exp. Cell Res., 256:248-256, 2000.

Salomoni, P., Conderelli, F., Sweeney, S.M., and Calabretta, B. Versatility of BCR/ABL-expressing leukemic cells in circumventing pro-apoptotic BAD effects. Blood, 96:676-684, 2000.

Perrotti, D., Iervolino, A., Cesi, V., Cirinna, M., Grassilli, E., Bonatti, S., Claudio, P.P. and Calabretta, B. BCR/ABL prevents c-Jun-mediated, proteasome-dependent and ubiquitin-independent TLS/FUS proteolysis through a PKCbll-dependent pathway. Mol. Cell. Biol., 20:6159-6169, 2000.

Dahéron, L. Zenz, T., Siracusa, L.D., Brenner, C., and Calabretta, B. Molecular cloning of lan4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity. Nucleic Acids Res., 29:1308-1316, 2001.

Condorelli, F., Salomoni, P., Cottoret, S., Srinivasula, S., Alnermi, E., and Calabretta, B.Caspase cleavage enhances the apoptosis-inducing effects of BAD. Mol. Cell. Biol., 21:3025-3036, 2001.

Powzaniuk, M., Trotta, R., Loza, M.J., Harth, A., Iozzo, R.V., Eisenlohr, L.C., Perussia, B., and Calabretta, B. B-Myb overexpression results in activation and increased Fas/FasL-mediated cytotoxicity of T and NK cells. J. Immunol., 167:242-249, 2001.

Perrotti, D., Cesi, V., Trotta, R., Guerzoni, C., Santilli, G., Campbell, K., Iervolino, A., Condorelli, F., Gambacorti-Passerini, C., Caligiuri, M.A. and Calabreta, B. BCR-ABL suppresses C/EBP a expression through inhibitory action of hnRNP E2. Nature Genet., in press.