Lisa S. Chen

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-X(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction.

Mechanisms of cytotoxicity to pim kinase inhibitor, SGI-1776, in acute myeloid leukemia

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound, SGI-1776 inhibits Pim-1, Pim-2 and Pim-3, and was evaluated in AML-cell line, -xenograft model, and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets, c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47), were both decreased in actively cycling AML cell lines MV-4-11, MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2, Bcl-x(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data, xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly, SGI-1776 was also cytotoxic in AML primary cells, irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment.

RNA-directed agent, cordycepin, induces cell death in multiple myeloma cells

Multiple myeloma (MM) is an incurable plasma cell malignancy that is slow‐growing, and thus traditional DNA‐replication directed chemotherapeutics are ineffective. We hypothesized that those agents that target RNA‐directed processes would be successful in MM. To test this postulate, cordycepin, a polyadenylation inhibitor was used as a proof‐of‐principle towards MM cell lines. Cordycepin accumulated in MM.1S cells as its triphosphate metabolite, 3′dATP and subsequently inhibits RNA synthesis and cell growth. Cell death was via apoptosis induction and over 50% of treated cells were annexin‐V positive after 48 h. As a consequence of RNA synthesis inhibition, we hypothesized that specific genes with short half‐lives may be downregulated, leading to a reduction in protein. Indeed, a reduction in the transcript levels for MET, a survival gene for MM, was detected as early as 4 h and transcripts were reduced to c. 10% of control after 48 h. Interestingly, no significant change in protein levels was observed for Bcl‐2, XIAP, Mcl‐1 or survivin. Stabilization of p53 was not observed, and caspases‐8, ‐9 and ‐3 showed activation following cordycepin treatment but were not required for cell death. Our results suggest that RNA‐directed agents may be a new group of agents for the treatment of MM.

Inflammation and survival pathways: Chronic lymphocytic leukemia as a model system

A primary response to inflammation is an increased survival of the target cell. Several pathways have been identified that promote maintenance of the cell. The principal mechanism for the extended survival is through induction of anti-apoptotic Bcl-2 family proteins. Bcl-2 was the founding member of this family with five additional members, Bcl-X(L), Bcl-W, Bcl-B, Bfl-1, and Mcl-1, discovered mostly in hematological malignancies. Another mechanism that could add to cell survival is the Pim kinase pathway. This family of enzymes is associated with Myc-driven transcription, cell cycle regulation, degradation of pro-apoptotic proteins, and protein translation. Chronic lymphocytic leukemia serves as an optimal model to understand the mechanism by which these two protein families provide survival advantage to cells. In addition, since this malignancy is known to be maintained by microenvironment milieu, this further adds advantage to investigate mechanisms by which these pro-survival proteins are induced in the presence of stromal support. Multiple mechanisms exists that result in increase in transcript and protein level of anti-apoptotic Bcl-2 family members. Following these inductions, post-translational modifications occur resulting in increased stability of pro-survival proteins, while Pim-mediated phosphorylation inhibits pro-apoptotic protein activity. Furthermore, there is a cross-talk between these two (Bcl-2 family proteins and Pim family proteins) pathways that co-operate with each other for CLL cell survival and maintenance. Vigorous efforts are being made to create small molecules that affect these proteins directly or indirectly. Several of these pharmacological inhibitors are in early clinical trials for patients with hematological malignancies.

Transcription and translation are primary targets of Pim kinase inhibitor, SGI-1776, in mantle cell lymphoma

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are serine/threonine/tyrosine kinases and oncoproteins that promote tumor progression. Three isoforms of Pim kinases have been identified and are known to phosphorylate numerous substrates, with regulatory functions in transcription, translation, cell cycle, and survival pathways. These kinases are involved in production, proliferation, and survival of normal B cells and are overexpressed in B-cell malignancies such as mantle cell lymphoma (MCL). SGI-1776 is a small molecule and Pim kinase inhibitor with selectivity for Pim-1. We hypothesize that Pim kinase function can be inhibited by SGI-1776 in MCL and that inhibition of phosphorylation of downstream substrates will disrupt transcriptional, translational, and cell cycle processes and promote cell death. SGI-1776 treatment in 4 MCL cell lines resulted in apoptosis induction. Phosphorylation of transcription (c-Myc) and translation targets (4E-BP1), tested in Jeko-1 and Mino, was declined. Consistent with these data, Mcl-1 and cyclin D1 protein levels were decreased. Importantly, similar to cell line data, MCL primary cells but not normal cells showed similar inhibition of substrate phosphorylation and cytotoxicity from SGI-1776 treatment. Genetic knockdown of Pim-1/Pim-2 affected similar proteins in MCL cell lines. Collectively these data demonstrate Pim kinases as therapeutic targets in MCL.

Mcl-1: the 1 in CLL

Although Mcl-1 has been established as a survival and maintenance protein during in vitro incubations of primary CLL cells, in this issue of Blood , Pepper and colleagues further demonstrate the association of Mcl-1 with other prognostic factors and the role of this antiapoptotic protein in disease

Biological Effects of the Pim Kinase Inhibitor, SGI-1776, in Multiple Myeloma

BACKGROUND Pim kinases are constitutively active serine/threonine/tyrosine kinases that are overexpressed in hematological malignancies such as multiple myeloma. Pim kinase substrates are involved in transcription, protein translation, cell proliferation, and apoptosis. SGI-1776 is a potent Pim kinase inhibitor that has proven to be cytotoxic to leukemia and lymphoma cells. Based on this background, we hypothesized that SGI-1776 treatment would result in myeloma cytotoxicity. MATERIALS AND METHODS To test this, myeloma cell lines and primary CD138(+) cells from myeloma patients were treated with SGI-1776 in a dose- and time-dependent manner, and effect on cell death and proliferation, induction of autophagy, and changes in cell cycle profile were measured. RESULTS SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138(+) cells (< 10%) assessed using Annexin-V/propidium iodide. Limited effect was observed in cell cycle profile or growth in cell lines. However, DNA synthesis was decreased by 70% at 3 μM (all time points) in U266 though this was not observed in MM.1S. In accordance, immunoblot analyses revealed no change in transcription (c-Myc and H3), or apoptotic (Bad) proteins that are substrates of Pim kinases. In contrast, autophagy, assessed using acridine orange staining, was induced with SGI-1776 treatment in both cell lines (U266, 25%-70%; MM.1S, 8%-52%) and CD138(+) cells (19%-21%). Immunoblot analyses of the autophagy LC3b marker and translation initiation proteins (phospho-p70S6K and 4E-BP1) corroborated autophagy induction. CONCLUSION These data indicate that SGI-1776 treatment in myeloma cell lines and CD138(+) myeloma cells elicits its deleterious effects through inhibition of translation and induction of autophagy.

Inhibition of ATP synthase by chlorinated adenosine analogue

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; F(O) intermembrane base and F1 domain, containing alpha and beta subunits. Crystal structures of both alpha and beta subunits that bind to the substrate, ADP, are known in tight binding (alpha(dp)beta(dp)) and loose binding (alpha(tp)beta(tp)) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the alpha(tp)beta(tp) state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (alpha(dp)beta(dp)) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF(3)(-). Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.

Impact of bone marrow stromal cells on Bcl-2 family members in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western world. High levels of Bcl-2 family anti-apoptotic proteins are responsible for apoptosis resistance. Besides anti-apoptotic proteins, the microenvironment provides substantial survival signals to CLL leukemic cells. However, in-depth knowledge on the role of individual Bcl-2 family members in the context of the microenvironment is still limited. We performed a comprehensive analysis of transcripts and proteins of 18 Bcl-2 family members using an “apoptosis array microfluidic card” in primary cells before and after stromal co-cultures. Our data showed that five of six anti-apoptotic members (excluding Bcl-b), two of three pro-apoptotic members (excluding Bok) and six of nine BH3-only members were present at detectable mRNA levels in CLL cells. Importantly, stromal-mediated extended survival of CLL cells was strongly associated with elevated global transcription. Upon co-culturing with stromal cells, there was an early response of an increase in anti- (2/5) and pro-apoptotic protein (3/8) transcripts on day 1, while an increase in anti-apoptotic proteins was observed on day 3, with no significant change in pro-apoptotic proteins. Our study revealed a differential pattern of expression of both transcripts and proteins following stromal co-cultures, proposing a significance of Bcl-2 family members in the stromal microenvironment.

Protein profiling identifies mTOR pathway modulation and cytostatic effects of Pim kinase inhibitor, AZD1208, in acute myeloid leukemia

Abstract Pim kinases phosphorylate and regulate a number of key acute myeloid leukemia (AML) cell survival proteins, and Pim inhibitors have recently entered clinical trial for hematological malignancies. AZD1208 is a small molecule pan-Pim kinase inhibitor and AZD1208 treatment resulted in growth inhibition and cell size reduction in AML cell lines including FLT3-WT (OCI-AML-3, KG-1a, and MOLM-16) and FLT3-ITD mutated (MOLM-13 and MV-4-11). There was limited apoptosis induction (<10% increase) in the AML cell lines evaluated with up to 3 μM AZD1208 for 24 h, suggesting that growth inhibition is not through apoptosis induction. Using reverse phase protein array (RPPA) and immunoblot analysis, we identified that AZD1208 resulted in suppression of mTOR signaling, including inhibition of protein phosphorylation of mTOR (Ser2448), p70S6K (Thr389), S6 (Ser235/236), and 4E-BP1 (Ser65). Consistent with mTOR inhibition, there was also a reduction in protein synthesis that correlated with cell size reduction and growth inhibition with AZD1208; our study provides insights into the mechanism of AZD1208.