ALBERT

Description: Acute promyelocytic leukemia (APL) is distinguished from other AMLs by cytogenetic, clinical, as well as biological characteristics. The hallmark of APL is the t(15;17) which leads to the expression of the PML/RAR fusion protein. PML/RAR is the central leukemia-inducing lesion in APL and is directly targeted by all trans retinoic acid (t-RA). Patients suffering from APL undergo complete hematologic but not molecular remission upon treatment with t-RA. Virtually all patients treated with t-RA-monotherapy had a rapid relapse within few months. But in the combination with an anthracycline, such as doxorubicin or idarubicin, t-RA improved the long term outcome of APL-patients dramatically. Nothing is known about why t-RA-monotherapy is unable to eradicate completely the leukemic population and how it increases the response to chemotherapy. In vitro, the exposure of early hemopoietic stem cells (HSCs) to t-RA does not induce differentiation but selects immature progenitors. Moreover, mice lacking the t-RA-specific receptor RARalpha do not exhibit an impairment of granulopoiesis or hemopoiesis. The indication, that t-RA may be involved in the hemopoietic differentiation, is given by the HL-60 cell line which undergoes granulocytic differentiation at the pharmacological dosages (10−6M) of t-RA. Furthermore vitamin A-deficient mice or mice treated with a antagonist of t-RA accumulate more immature granulocytes in the bone marrow. PML/RAR mediates the response of APL blasts to t-RA, but it is completely unclear, which effect t-RA exerts on the PML/RAR-positive leukemic stem cells which maintains the blast population and represents the source of relapse. Therefore we investigated the effect of t-RA on a cell population with stem cell capacity expressing PML/RAR isolated from the APL cell line NB4 as well as from CD34+/CD38- KG-1 cells transfected with PML/RAR. Here we report that i) the NB4 cells engrafted in NOD/SCID mice indicating the presence of a subpopulation with stem cell capacity in NB4 cells; ii) NB4 had a Hoechst 3342 excluding side population (SP) representing about 1% of the whole cell population; iii) t-RA reduced but did not deplete the side population in NB4 cells; iv) the expression of PML/RAR increased CD34+/CD38- population in KG-1 cells from 75% to over 95%; v) t-RA reduced the CD34+/CD38- population from 75% to 3,5% in mock transfected KG-1 confirming its capacity to induce differentiation, whereas in PML/RAR-positive KG-1 cells it led only to a reduction from 98% to a 25%, which still maintain the capacity to engraft in NOD-SCID mice; vi) also the expression of other fusion proteins, such as AML-1/ETO or PLZF/RAR, associated with t-RA-resistant AML-subtypes, increased the percentage of CD34+/CD38- KG-1 cells over 90%, which was reduced by t-RA only to 35% and 19%, respectively. Taken together these data suggest that a subset of early HSC expressing PML/RAR exhibit the same t-RA-resistant phenotype as HSC expressing fusion proteins associated with AML-subtypes which, in contrast to APL, do not respond to t-RA. These data may give an explanation, why APL-patients do not achieve complete molecular remission upon t-RA monotherapy and undergo early relapse.

Description: In 95% of chronic myeloid leukemia (CML) and in 25% of acute lymphatic leukemia (ALL) the t(9;22) translocation fuses the bcr gene on chromosome 22 to the abl gene on chromosome 9 and vice versa. On 22+ the different breakpoints leads to the formation of two different major fusion genes: the major breakpoint (M-bcr) related to CML and the minor (m-bcr) related to ALL. The chimaeric fusion gene on 22+ (Philadelphia-chromosome) encodes for the BCR/ABL protein, the p210(BCR/ABL) in CML and the p185(BCR/ABL) in Ph+ALL. The fusion gene on 9+ encodes for the reciprocal ABL/BCR proteins, the p40(ABL/BCR) in CML and the p96(ABL/BCR) in Ph+ALL. The respective ABL/BCR transcripts are detectable in 65% of CML and 100% of Ph+ ALL patients. The ABL/BCRs are BCR mutants and thus N-terminally truncated Rho-guanine-nucleotide exchange factors (Rho-GEF’s). It is known that the N-terminal truncation can confer transformation potential to Rho-GEFs, such as NET-1. In addition, both ABL/BCRs, like wt BCR, contain a C-terminal Rac-GTPase activating protein (GAP)-domain. CML-associated ABL/BCR (p96(ABL/BCR)) differs from the Ph+ ALL-associated p40(ABL/BCR) in that that it misses the ‘dbl homology domain’(DH domains) of potential oncogenic function. Hence it seems that Ph+ALL blasts, in contrast to CML-blasts, express, as a consequence of t(9;22) translocation, two oncogenic fusion proteins, the p185(BCR-ABL) as well as the p40(ABL/BCR) protein. Actually nothing is known about the contribution of the reciprocal t(9;22) translocation products to the CML- and the ALL-phenotype. Thus we studied the phenotype induced by the ABL/BCRs in hemopoietic progenitors. Here we report that both ABL/BCRs i) lost the capacity of wt BCR to suppress the activation of RAC by its Rac-GAP domain, but did not influence the activation status of Rho or cdc42; ii) as a consequence of the deregulation of Rac the cytoskeleton modelling by BCR (Filopodia - cdc42-like phenotype) was altered in p40(ABL/BCR)- and p96(ABL/BCR)-expressing fibroblasts (stress fibers - Rho-like phenotype and “microspikes”, respectively); iii) the increase of migration of BCR-expressing 32D cells into a stroma cell-spheroid model was reverted in p40(ABL/BCR)- and p96(ABL/BCR)-expressing 32D cells; iv) adhesion to TNFalpha activated endothelial cell layer in the “flow chamber” was increased in BCR-positive 32D cells but not in p40(ABL/BCR)- and p96(ABL/BCR)-positive cells. Regarding their leukemogenic potential we showed that i) both ABL/BCRs, in contrast to wt BCR, activated RAS; ii) both ABL/BCRs were unable to transform fibroblasts and to render Ba/F3 cells factor-independent. iii) p96(ABL/BCR) increased the replating efficiency of Sca1+/lin- hemopoietic stem cells (HSC) by selecting a population of immature HSC exclusively expressing c-kit and Sca-1 more strongly than p40(ABL/BCR); iv.) both ABL/BCR blocked the myeloid differentiation of HSC v) the inoculation of p96(ABL/BCR)- or p40(ABL/BCR)-expressing HSC into lethally irradiated recipient mice led in the 40% and 60% of the cases, respectively, to a clinical picture of either acute leukemia or myeloproliferative syndrome within 2–9 months. These data show for the first time that the t(9;22) leads to two leukemogenic fusion proteins - the BCR/ABL and the ABL/BCR - in CML as well as in Ph+ALL, which might represent an additional target for molecular therapy approaches.

Description: Histone deacetylase inhibitors (HDI) have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts either alone, or in combination with differentiating agents such as all-trans retinoic acid (ATRA). We have previously reported favorable effects of the potent HDI valproic acid (VPA) in combination with ATRA in a small subset of patients with advanced acute myeloid leukemia (AML) leading to blast cell reduction and improvement of hemoglobin. This effect was accompanied by hypergranulocytosis most likely due to an enhancement of non-leukemic myelopoiesis and suppression of malignant hematopoiesis rather than enforced differentiation of leukemic cells. These data prompted us to investigate the impact of VPA on normal hematopoietic stem cells (HSC). Differentiation of cord blood-derived, purified CD34+ cells was assessed by FACS analysis after a 7-days suspension culture in presence of early acting cytokines and 30–150μg/mL VPA. VPA prevented differentation of CD34+ cells in a dose-dependent manner: concomitant with an increase of CD34+ cells from 17 to 47%, the proportion of monocytic CD14+ cells decreased from 27 to 3% (n=3). In addition, VPA induced a 30-fold amplification of CD34+ bone marrow (BM) cells within 10 days as determined by colony assays (n=3). To evaluate the functional capacity of VPA-treated HSC, murine Sca1+/lin−s cells were harvested from colony assays and replated. VPA treatment allowed up to four cycles of replating in contrast to VPA-naïve control cells. Further analysis demonstrated that the stimulatory effect of VPA on the in vitro growth and colony formation capacity of HSC was mainly due to accelerated cell cycle progression. VPA strongly increased the proportion of cells in S phase compared to untreated controls (38 vs. 17%, resp.), as detected by propidium iodid staining and BRDU incorporation as well as reduced expression of the CDK-inhibitor p21cip-1/waf using murine HSC after 7 days of culture. Downregulation of p21cip-1/waf was confirmed in CD34+ BM cells showing maximum inhibition after 48 hours of VPA treatment and no recovery thereafter. Recent results indicate that VPA exerts inhibitory activity on GSK3beta by phosphorylation on Ser-9 and stimulates Akt in human neuroblastoma cells. GSK3beta is an effector of the Wnt-signaling pathway located upstream of beta-catenin. Wnt-signaling can directly stimulate the proliferation of HSC, expand the HSC pool and lead to upregulation of HoxB4. Here we show that VPA increased the inhibition-associated phosphorylation of GSK3beta on Ser-9 in human CD34+ BM cells after 48 hours as well as in murine Sca1+/lin− cells after 7 days. Exposure to VPA enhanced beta-catenin and Akt activity not only in CD34+ HSC but also in KG-1 and TF-1 cells with maximum activation after 48 hours of VPA stimulation. Moreover, VPA lead to an 8-fold increase of the HoxB4 level in CD34+ BM cells as determined by real time PCR at 48 hours. In conclusion, we show that VPA i.) expands HSC as assesed by phenotype and function; ii.) accelerates cell cycle progression of HSC accompanied by the down-regulation of p21cip-1waf; iii.) activates the GSK3beta depending beta-catenin pathway and Akt and iv.) up-regulates HoxB4. Our data strongly suggest that VPA is able to influence some of the signaling pathway considered relevant for proliferation and self-renewal which might request reconsideration of their employment for the treatment of AML.

Description: Translocations involving the abl locus on chromosome 9 fuses the tyrosine kinase c-ABL to proteins harboring oligomerization interfaces such as BCR or TEL, enabling these ABL-fusion proteins (X-ABL) to transform cells and to induce leukemia. The ABL kinase activity is blocked by the ABL kinase inhibitor STI571 which abrogates transformation by X-ABL. To investigate the role of oligomerization for the transformation potential of X-ABL and for the sensitivity to STI571, we constructed ABL chimeras with oligomerization interfaces of proteins involved in leukemia-associated translocations such as BCR, TEL, PML, and PLZF. We assessed the capacity of these chimeras to form high molecular weight (HMW) complexes as compared with p185(BCR-ABL). There was a direct relationship between the size of HMW complexes formed by these chimeras and their capacity to induce factor independence in Ba/F3 cells, whereas there was an inverse relationship between the size of the HMW complexes and the sensitivity to STI571. The targeting of the oligomerization interface of p185(BCR-ABL) by a peptide representing the coiled coil region of BCR reduced its potential to transform fibroblasts and increased sensitivity to STI571. Our results indicate that targeting of the oligomerization interfaces of the X-ABL enhances the effects of STI571 in the treatment of leukemia caused by X-ABL.

Description: The transcription factor Stat5 mediates the cellular response to activation of multiple cytokine receptors involved in the regulation of proliferation and differentiation of hematopoietic cells. Recently, the human Stat5 gene was found to be translocated to the RARα gene in a patient with acute promyelocytic leukemia indicating that Stat5 might also play a role in cellular transformation. We investigated the mechanism by which Stat5 might exert this function and studied the biochemical and cellular functions of fusion proteins comprising Stat5 and RARα. The expression of Stat5-RARα causes the transcriptional repression of gene transcription, a process that requires the coiled-coil domain of Stat5 (amino acid positions 133-333). Oligomerization of this domain in the Stat5-RARα fusion protein leads to stable binding of the corepressor SMRT independent of all-trans retinoic acid (ATRA) stimulation and is accompanied by an impaired response to differentiation signals in hematopoietic cells. This inhibitory effect on myeloid differentiation cannot be overcome by simultaneous coexpression of RARα. We conclude that Stat5 is capable of interacting with a corepressor complex that alters the pattern of corepressor binding to RARα and its dissociation in response to ATRA stimulation, leading to enhanced repressor activity and a block of hematopoietic differentiation.