Characterization of proteins binding the 3′ regulatory region of the IL-3 gene in IL-3-dependent and autocrine-transformed hematopoietic cells

Previously we documented the prolongation of the IL-3 mRNA half-life in an autocrine-transformed cell line. This cell line has an intracisternal type A particle transposition in the IL-3 mRNA 3′ untranslated region which displaced four out of six AUUUA motifs involved in IL-3 mRNA destabilization. In this study, the proteins binding to the IL-3 mRNA AU-rich elements (ARE) were examined. Specific protein binding was detected to the wild-type IL-3 ARE region which contained 6 AUUUA motifs (AU6). In contrast, no binding was detected to the mutated IL-3 ARE region which contained only two AUUUA motifs (AU2). Proteins with apparent molecular weights of 36, 40, 43, 46, 55, 57, 68 and 95 kDa were bound to AU6 motif. The hnRNP C and AUF-1 (hnRNP D) proteins were determined to be two of the IL-3 ARE binding proteins. Incubation of protein extracts with antibodies to hnRNP C and AUF-1 significantly decreased the protein binding to the IL-3 ARE. Treatment of IL-3 dependent cells with calcium ionophores eliminated the proteins binding to the ARE in wild-type IL-3-dependent FL5.12 cells and also resulted in the accumulation of IL-3 mRNA transcripts with a long half-life. These results indicated that there was a specific complex which bound the IL-3 mRNA 3′ ARE. Mutations which truncate the IL-3 ARE eliminate the ability of proteins to bind this regulatory region and can result in autocrine transformation due to the presence of IL-3 mRNA transcripts with a long half-life

Co-targeting of Bcl-2 and mTOR pathway triggers synergistic apoptosis in BH3 mimetics resistant acute lymphoblastic leukemia

Several chemo-resistance mechanisms including the Bcl-2 protein family overexpression and constitutive activation of the PI3K/Akt/mTOR signaling have been documented in acute lymphoblastic leukemia (ALL), encouraging targeted approaches to circumvent this clinical problem. Here we analyzed the activity of the BH3 mimetic ABT-737 in ALL, exploring the synergistic effects with the mTOR inhibitor CCI-779 on ABT-737 resistant cells. We showed that a low Mcl-1/Bcl-2 plus Bcl-xL protein ratio determined ABT-737 responsiveness. ABT-737 exposure further decreased Mcl-1, inducing apoptosis on sensitive models and primary samples, while not affecting resistant cells. Co-inhibition of Bcl-2 and the mTOR pathway resulted cytotoxic on ABT-737 resistant models, by downregulating mTORC1 activity and Mcl-1 in a proteasome-independent manner. Although Mcl-1 seemed to be critical, ectopic modulation did not correlate with apoptosis changes. Importantly, dual targeting proved effective on ABT-737 resistant samples, showing additive/synergistic effects. Together, our results show the efficacy of BH3 mimetics as single agent in the majority of the ALL samples and demonstrate that resistance to ABT-737 mostly correlated with Mcl-1 overexpression. Co-targeting of the Bcl-2 protein family and mTOR pathway enhanced drug-induced cytotoxicity by suppressing Mcl-1, providing a novel therapeutic approach to overcome BH3 mimetics resistance in ALL

Therapeutic targeting of CK2 in acute and chronic leukemias

CK2 is a ubiquitously expressed, constitutively active Ser/Thr protein kinase, which is considered the most pleiotropic protein kinase in the human kinome. Such a pleiotropy explains the involvement of CK2 in many cellular events. However, its predominant roles are stimulation of cell growth and prevention of apoptosis. High levels of CK2 messenger RNA and protein are associated with CK2 pathological functions in human cancers. Over the last decade, basic and translational studies have provided evidence of CK2 as a pivotal molecule driving the growth of different blood malignancies. CK2 overexpression has been demonstrated in nearly all the types of hematological cancers, including acute and chronic leukemias, where CK2 is a key regulator of signaling networks critical for cell proliferation, survival and drug resistance. The findings that emerged from these studies suggest that CK2 could be a valuable therapeutic target in leukemias and supported the initiation of clinical trials using CK2 antagonists. In this review, we summarize the recent advances on the understanding of the signaling pathways involved in CK2 inhibition-mediated effects with a particular emphasis on the combinatorial use of CK2 inhibitors as novel therapeutic strategies for treating both acute and chronic leukemia patients

Combined effects of aberrant MEK1 activity and BCL2 overexpression on relieving the cytokine dependency of human and murine hematopoietic cells

The MEK1 oncoprotein plays a critical role in Ras/Raf/ MEK/MAPK-mediated transmission of mitogenic signals from cell surface receptors to the nucleus. In order to examine this pathway's role in leukemic transformation, a conditionally active (β-estradiol-inducible) form of the MEK1 protein was created by ligating a cDNA encoding an N-terminal truncated form of MEK1 to the hormone-binding domain of the estrogen receptor (ER). We introduced this chimeric ΔMEK1:ER oncoprotein into cytokine-dependent human TF-1 and murine FDC-P1 hematopoietic cell lines. Two different types of cells were recovered after drug selection in medium containing either cytokine or β-estradiol: (1) cells that expressed the ΔMEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to ΔMEK1:ER activation. Cytokine-dependent cells were recovered 102 to 104 times more frequently than MEK1-responsive cells depending upon the particular cell line. To determine whether BCL2 overexpression could synergize with the ΔMEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent ΔMEK1:ER-expressing cells were infected with a BCL2-containing retrovirus, and the frequency of MEK1-responsive cells determined. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cells, however, it did increase the frequency at which MEK1-responsive cells were recovered approximately 10-fold. ΔMEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily lost upon withdrawal of β-estradiol in the MEK1-responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to ΔMEK1:ER stimulation in both ΔMEK1:ER and ΔMEK1:ER+BCL2 cells. As compared to the cytokine-dependent ΔMEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2. While both MEK1-responsive ΔMEK1:ER and ΔMEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the MEK1-responsive ΔMEK1:ER+BCL2 cells than in the MEK1-responsive cells lacking BCL2 or cytokine-dependent cells. These conditionally transformed cells will be useful in furthering our understanding of the roles MEK1 and BCL2 play in the prevention of apoptosis in hematopoietic cells

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G0/G1 phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3a/b and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34þ/CD4/CD7), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL

AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications.

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be downmodulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4þ T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34þ/CD7/CD4) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL. Leukemia (2012) 26, 91–100; doi:10.1038/leu.2011.269; published online 4 October 201