ALBERT

Description: Activation of double-stranded RNA-activated Protein Kinase (PKR) is associated with growth inhibition and cell death. PKR is normally dormant in cells and is activated in response to stress challenge (e.g. viral infection, chemotherapy, factor withdrawal). The presence of active kinase under growth conditions is generally not observed. PKR expression and activation status was determined in four ALL cell lines: REH, CCRF-CEM, MOLT4, and RS(4;11). PKR was found to be expressed and phosphorylated in all the cell lines. The presence of active PKR suggests the kinase may be important for cellular homeostasis in ALL cells. The best characterized substrate of PKR is eIF2α. Phosphorylation of eIF2α was examined to determine if PKR phosphorylation levels correlated with phosphorylation of the kinase’s primary substrate. Phosphorylated eIF2α was detected in all the cell lines except RS(4;11) cells. The lack of observed phosphorylated eIF2α in the RS(4;11) cells was due to the low abundance of the eIF2α protein. Another substrate of PKR is B56α, a B regulatory subunit of Protein Phosphatase 2A (PP2A). B56α comprises a mitochondrial PP2A isoform that serves as the BCL2 phosphatase and promotes cell death in response to chemotherapeutic agents. PKR was found to phosphorylate B56α at serine 28 in REH cells to promote mitochondrial PP2A activity and BCL2 phosphatase function. Loss of PKR by shRNA results in phosphorylation of BCL2 and the cells become resistant to the chemotherapeutic drug etoposide. Inhibition of PKR by pharmacologic inhibitor or shRNA in REH cells results in loss of B56α expression suggesting that phosphorylation may be required for stability of the B subunit. However, phosphorylation of B56α is not required for its stability since mutant S28A B56α protein is readily expressed in cells. A clue to an alternative mechanism was provided by the eIF2α phosphorylation status and B56α expression pattern in the four ALL cell lines. RS(4;11) cells display little if any B56α protein while the other three ALL cell lines express the B subunit. REH, CCRF-CEM, and MOLT4 cells exhibited ~ 3X higher mitochondrial PP2A activity compared to RS(4;11) cells. The lack of mitochondrial PP2A activity in RS(4;11) cells is likely due to a lack of B56α. Since RS(4;11) cells have active PKR but lack phosphorylated eIF2α, the possibility arises that B56α expression may depend on phosphorylated eIF2α. The mechanism how PKR promotes B56α expression appears to involve the proteasome since a proteasome inhibitor can block loss of the B subunit when PKR is suppressed. A role for eIF2α is suggested since salubrinal, a drug that promotes eIF2α phosphorylation by preventing the dephosphorylation of the molecule increased expression of B56α protein. It has recently been found that eIF2α phosphorylation can activate the PI3K/AKT signaling cascade. Suppression of PKR in REH cells blocks AKT phosphorylation suggesting that PKR may positively regulate AKT in these cells. It appears that the mechanism how PKR promotes B56α expression involves the PI3K/AKT cascade since inhibition of this signaling pathway using LY294002 blocks expression of the B subunit. Since B56α mediated mitochondrial PP2A activity promotes cell death in response to chemotherapeutic agents, it is likely that the normally pro-survival PI3K/AKT pathway may serve a role in stress signaling in ALL cells by supporting B56α expression to promote BCL2 phosphatase activity. These findings indicate that PKR can regulate the BCL2 phosphatase at multiple levels in ALL cells. Understanding the complex regulatory pathway how PKR controls PP2A and BCL2 will be necessary for designing effective therapies for the treatment of ALL.

Description: The bone marrow microenvironment (BME) critically supports hematopoietic stem cells and protects leukemia cells from chemotherapy, immune surveillance, and related stresses. A critical component of the BME is the mesenchymal stem cell (MSC). Dan Link’s group demonstrated that MSC are essential for human hematopoiesis, particularly as a source of SDF-1, which regulates homing, proliferation, and differentiation of HSC. Moreover, studies from our group and others have demonstrated that MSC protect leukemia cells from chemotherapy. At present, very little is known about MSC derived from AML patients, and an understanding of the proteomic makeup of these cells in the leukemia microenvironment could help to elucidate mechanisms involved in supporting their pro-tumor function. We used reverse phase protein array analysis (RPPA) to compare the expression of 151 proteins in MSC derived from AML BMs (N = 106) with those from healthy donors (N = 71). The expression of 45 of these proteins was deemed significantly different (p 〈 0.01) between the two sets. AML MSC expressed higher levels of p53 and p21 (CDKN1A), and the expression of the latter was correlated with other proteins within each MSC set. Using beta-galactosidase staining, AML MSC were found to undergo senescence more frequently than normal MSC. Elevated p21 in AML MSC is consistent with this finding. While 15 proteins were positively, and 20 proteins negatively, correlated with p21 expression in normal MSC, there were only three proteins positively, and nine negatively, correlated in AML-derived MSC. In normal MSC, SMAD1 (a key component in MSC growth and differentiation involving multiple receptors like TGF beta and BMP) expression and AKT signaling were low when p21 is expressed. However, in AML MSC this association was not seen, albeit a negative correlation with ITGAL was observed. SMAD1 expression was higher in normal MSC. In normal MSC, the expression of SMAD1 was negatively correlated with PPARG and NPM1, and was positively correlated with the expression of phosphorylated ELK. The opposite relationship was seen in AML MSC (i.e., PPARG and NPM1 exhibited positive correlation with SMAD1 and phosphorylated ELK was negatively correlated with the protein). While the significance of these relationships remains to be determined it is interesting to note that PPARG is a key regulator of adipocyte differentiation in MSC, so perhaps this alteration of SMAD/PPARG in AML MSC could impede their differentiation potential. In an accompanying abstract from our group, we report that AML MSC are primed toward osteoblastic differentiation and do not differentiate into adipocytes (Battula VL et al, ASH 2014). The RPPA data on PPARG is consistent with this finding. SMAD1 also positively regulates miR-21. Since p21 is a miR-21 target, it seems possible that the differences in expression could be attributed to SMAD1 and miR-21 signaling. We analyzed miR-21 expression in normal and AML-derived MSC (N = 10, each) using qRT-PCR and found a statistically significant (p =0.014) increase in its expression in normal MSC relative to their disease counterparts. When anti-miR-21 was transduced into healthy donor MSC, which caused a 3-fold increase in p21 (but no difference in cyclin D1 expression, another miR-21 target whose expression was also increased in AML MSC). AML MSC also exhibited higher protein expression of the B55 alpha subunit (PPP2R2A) of protein phosphatase 2A (PP2A). This expression contrasted interestingly with that of leukemia cells, since we have previously reported low PPP2R2A levels in AML blasts associated with shorter remission durations (Ruvolo et al Leukemia 2011). Furthermore, AKT phosphorylation was negatively correlated with PPP2R2A expression in AML blasts, and normal MSC, but there was no correlation between PPP2R2A and phosphorylated AKT in AML MSC. Also, expression of PPP2R2A was positively correlated with the expression of the survival protein NOL3 (ARC) which may provide new clues to possible survival mechanisms in AML MSC. In summary, these findings represent insights into the proteomic profiling of normal and AML MSC. Results suggest that senescence (via p21), differentiation potential (involving SMAD/PPARG pathway), and survival signaling (including PP2A/AKT) are altered in AML MSC. Studies are underway to determine how these variations in MSC properties impact the AML microenvironment. Disclosures Carter: Tetralogic Pharmaceuticals: Research Funding.

Description: Apoptosis inducing factor (AIF) is a mitochondrial flavoprotein that is released into the cytoplasm to form an active DNase and then translocates into the nucleus to induce DNA fragmentation when cells undergo apoptosis. AIF also acts as an essential NADPH oxidase, a function which is distinct from its proapoptotic role. We have shown previously that AIF expression level increased with increasing blast counts in primary acute myelogenous leukemia (AML) patient samples. Here we report that AIF expression levels are increased in primary CD34+ AML cells compared with CD34− cells. To investigate the role of AIF in primary AML, bone marrow or peripheral blood samples from AML patients (n=8) with CD34+ or CD34− cell populations were examined for changes in reactive oxygen species (ROS), mitochondrial membrane potential (ΔψM), apoptosis and chemo-sensitivity in the CD34+ and CD34− compartments after treatment with the mitochondrial complex 1 inhibitor rotenone. Similar experiments were performed following knockdown of AIF with lentiviral shRNA directed against AIF in K562 cells. Results demonstrate: significantly increased apoptosis rate (39.2% Annexin V+ cells in CD34+ vs. 14.1% in CD34− cells following 2 uM rotenone, p=0.05), and decreased ΔψM in CD34+ cells (44.5% ΔψM cells in CD34+ vs. 78.2% in CD34− cells, p=0.01). MCL-1 expression levels were significantly decreased in CD34+ but not in CD34− cells (p=0.007). The apoptotic effect of rotenone in CD34+ cells was potentiated by AraC (1uM). Profound inhibition of O2 consumption in AIF knockdown K562 cells (−5.2±0.3) compared with K562-neo (neo transfected K562, −13.1±0.3) cells (p=0.001). Rotenone treated AIF knockdown K562 cells and K562-neo cells revealed that decreased AIF expression led to higher ΔψM and higher superoxide generation (p=0.0002). In contrast, total mitochondrial mass (NAO) decreased slightly in K562KD cells. Data suggested that AIF contributes to leukemia cell survival by modulating mitochondrial metabolism, independent of its proapoptotic activity, which is consistent with recent reports demonstrating its importance in mitochondrial complex 1 activity. Decreased AIF levels are associated with increased generation of superoxide anion, suggesting that AIF critically regulates mitochondrial function in AML cells. Rotenone selectively decreases AIF and induces apoptosis in CD34+ cells in AML. AIF functions as a survival factor in AML strategies targeting AIF expression or function may be beneficial in the treatment of myeloid leukemia.

Description: We have reported that “apoptosis repressor with caspase recruitment domain” (ARC), an antiapoptotic protein promotes leukemia-stromal interactions in part by increasing the expression of CXCR4 in AML cells and of CXCL12 (SDF-1) in mesenchymal stromal cells (MSCs) (Carter et al., ASH 2012). To further understand the mechanisms of action of ARC in mediating leukemia-stromal interactions, we examined the expression of multiple chemokines by quantitative cytokine PCR array in ARC knockdown (KD) and control MSCs. We found that among 6 C-X-C motif and 22 C-C motif chemokines, CXCL12, CCL2 and CCL4 are highly expressed and upregulated by ARC in MSCs. We then determined the protein levels of CCR2 and CCR5, the respective receptors for CCL2 and CCL4, in bone marrow samples from 8 AML patients and found that CCR2 was highly expressed in all samples and CCR5 was detectable in 75 % suggesting that the CCR2/CCL2 and CCR5/CCL4 axes may also contribute to leukemia-stromal interactions in AML. Trans-well migration assays showed that CCL2 and CCL4 induced the migration of AML cells which was antagonized by blocking antibodies or small molecule inhibitors. AML cells migrated less to ARC KD than to control MSCs. Primary leukemia cells from AML patient samples also migrated towards CCL2 and CCL4, and this migration correlated with the expression of the respective receptors in the leukemia cells. Co-culture of MSCs with leukemia cells greatly increased the levels of CXCL12, CCL2, and CCL4 in MSCs and the increase was diminished when co-cultured with ARC KD and increased when co-cultured with ARC overexpressing (OE) cells was compared to their respective controls, suggesting that chemokine expression in MSCs is in part regulated by ARC-mediated signaling from AML cells. Cytokine PCR array revealed that IL1β expression is increased in ARC OE and decreased in ARC KD cells. Conversely, the level of the IL1 receptor antagonist IL1RN is lower in ARC OE and higher in ARC KD. ARC OE cells also secreted more IL1β while ARC KD cells secreted less. Furthermore, co-cultures with MSCs increased the expression of IL1β in AML cells. IL1β greatly increased the levels of CCL2, CCL4, and CXCL12 in MSCs and the increase was largely diminished by co-treating the MSCs with IL1β antagonist IL1βRA. Similarly, treating MSCs with conditioned media from AML cells increased the expression of the chemokines in MSCs, and the increase was reduced with conditioned medium from ARC KD cells; this increase was diminished by IL1βRA. Furthermore, IL1βRA blocked the migration of these cells toward CCL2 and CCL4. Using a 3-D model in which cancellous bone was covered with MSCs, we found fewer OCI-AML3 cells attached to MSCs when ARC was knocked down in these cells further supporting the role of ARC in leukemia-stromal interactions. ARC is a member of caspase recruitment domain (CARD) containing proteins that have diverse functions such as antiapoptosis and regulation of NFκB activity. ARC participation in cell death suppression and leukemia-stromal interactions suggests that ARC may interact not only with apoptosis regulators but also with signaling proteins to regulate multiple cellular functions in AML. Conclusions Our findings suggest that multiple ARC-regulated receptor/ligand pairs play important roles in leukemia-stromal interactions, and that there is reciprocal crosstalk between malignant cells and microenvironment cells that is in part mediated through ARC-regulated inflammatory cytokine IL1β. ARC is therefore a novel target not only for direct apoptosis induction in leukemia cells but also for disruption of protective leukemia-stromal interactions to further sensitize leukemia cells to their elimination by chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: ONC201 (TIC10) is a novel small molecule that induces TRAIL-dependent apoptosis in various cancer cell types and is under development to enter a first-in-man study in advanced cancer patients .It was identified in a screen for small molecules capable of up-regulating endogenous TRAIL gene transcription in a p53-independent manner (Allen JE et al, Sci Transl Med., 2013). ONC201 triggers FOXO3a activation through dual inhibition of ERK and AKT, which transcriptionally upregulates TRAIL and TNFRSF10B (TRAIL-R2/DR5) in solid tumors. Because PI3K/AKT and MEK/ERK activation have been shown to be major contributors to drug resistance, ONC201 is potentially promising since it not only promotes TRAIL activation, but also upregulates its pro-apoptotic receptor DR5. Here we report the anti-lymphoma effects of ONC201 in MCL, a presently incurable disease. We treated three human MCL cell lines with wild-type p53 (Z-138, JVM-2, and Granta-519) and two similar lines with mutant p53 (MINO and Jeko-1) with ONC201. A 72-hour ONC201 treatment induced apoptosis in all MCL cell lines. Surprisingly, the p53 mutant MINO and Jeko-1 cells were more susceptible in apoptosis assays to ONC201 than cells with wild-type p53 (Fig.1) The effective concentrations inducing cell killing (as measured by annexin V positivity) in 50%/75% of the cells in the Z-138, JVM-2, MINO, Jeko-1, and Granta-519 cells were 9.9/〉10, 〉10/〉10, 2.6/5.2, 2.7/4.6 and 〉10/ 〉10 micromolar, respectively. We also treated five primary human MCL samples (three with wild-type p53 and two with mutant p53), and found that one of the two mutant p53 samples was highly sensitive to ONC201 as were the three samples with wild-type p53. One mutant p53 sample that was less sensitive to ONC201, was also resistant to Nutlin-3a and Ibrutinib suggesting an extremely drug-resistant phenotype. Real-time PCR analysis revealed that both DR5 and TRAIL mRNAs were transcriptionally upregulated in the primary MCL samples (a relative ratio of 7.25 compared to 3.13 in controls) after 72-hour treatment with ONC201. To determine the significance of p53 functional status in ONC201-induced apoptosis, p53 wild-type Z-138 and JVM-2 cells were stably transduced with lentivirus encoding either negative control shRNA or p53-specific shRNA and were exposed to ONC201 and results demonstrated complete p53-independence. Normal human bone marrow cells and mesenchymal stem cells were completely resistant to the cytotoxic effects of ONC201, which illustrated this agent's low toxicity against normal tissues. In order to examine the role of p53 activation in ONC201-induced apoptosis in MCL cells, we combined ONC201 with the MDM2 inhibitor Nutlin-3a. The combination cytotoxic effects of this combination were synergistic in p53 wild-type Z-138 and JVM-2 cells (combination index 0.87 and 0.63, respectively). Similar synergistic effects of ONC201 combined with the BTK inhibitor Ibrutinib were observed in Z-138 and MINO cells (combination index 0.63 and 0.61, respectively). This combination also triggered synergistic apoptotic effects in two primary MCL samples with combination indexes of 0.0011 and 0.073, respectively. Conclusion ONC201 induces p53-independent apoptosis in MCL cells, and may have significant clinical impact by targeting both p53 wild type and p53 mutant drug-resistant MCL cells. ONC201 exerts synergistic effects with MDM2 and BTK inhibitors that may be explored clinically. Disclosures: Allen: Drug Company: Employment. Andreeff:Oncoceutics: SAB Other.

Description: Galectins are a family of b-galactoside binding proteins with effects on cell adhesion, apoptosis, cell cycle, and mRNA processing. Galectin-3 (LGALS3) is unique among galectins by having an N terminal region of roughly 130 amino acids that allows for multimerization and binding to other proteins independent of carbohydrate binding. In addition to promoting BCL2 gene expression and mitochondrial integrity, LGALS3 (along with LGALS1) positively regulates RAS signaling and thus stabilizes survival proteins dependent on ERK phosphorylation such as MCL-1. The pro-survival functions of LGALS3 and other galectins suggest that their targeting could be therapeutic for cancers including AML. Indeed, LGALS3 expression is a predictor of poor prognosis in acute myeloid leukemia (AML), as reported by Cheng and colleagues (Blood 2013) for patients with non-M3 AML and CN-AML. The modified pectin GCS-100 (La Jolla Pharmaceutical, San Diego, CA), now in a Phase II clinical trial for chronic kidney disease, binds and blocks the function of LGALS3. We report that GCS-100 suppresses the growth of AML cell lines OCI-AML3, THP-1, and HL60 in vitro as a single agent, at doses under the 250 ug/mL (i.e., within clinically-achievable concentrations). Short-term treatment of cells (i.e., 〈 6 hr) potently suppressed phosphorylation of AKT and ERK and reduced expression of BCL2 and MCL-1. Because LGALS3 positively regulates anti-apoptotic BCL2 family members, the Raz group has suggested targeting galectins to enhance efficacy of BH3 mimetic drugs (Harazano et al Cancer Metastasis Review 2013). We found that GCS-100 potently synergized with ABT-737 to kill OCI-AML3 cells: while 1 uM ABT-737 or 125 ug/mL GCS-100 reduced total viable cells by ~ 30% and induced apoptosis in 〈 20% of cells after 48 hr as single agents, their combination at those doses and time point reduced viable cells by ~ 94% and induced apoptosis in ~ 70% of cells. Suppression of LGALS3 by lentiviral shRNA reduced BCL2 gene expression as determined by qRT-PCR and augmented killing with ABT-737. Lentiviral suppression of LGALS3 protected cells from GCS-100 at doses of 250 ug/mL but reduction of the galectin failed to protect cells from higher doses of the drug (i.e., 500 ug/mL). This result suggests other galectins are likely inhibited at higher doses of the agent. We used gene expression profiling (GEP) on Illumina HT12v4 human whole-genome arrays to assess more broadly the molecular effects of inhibiting galectins in AML cell lines OCI-AML3 and THP-1 treated with 250 ug/mL or 500 ug/ml GCS-100 for 24 hr. Data were analyzed by Gene Set Enrichment Analysis (GSEA) using gene sets from the Molecular Signatures Database (www.broadinstitute.org/gsea/msigdb/). GSEA suggested that GCS-100 promotes differentiation and inhibits genes associated with proliferation. Multiple upregulated gene sets suggest that there may be a release of a differentiation block as a result of GCS-100 treatment. Furthermore, two gene sets suggest that GCS-100 behaves similar to a GSK3 inhibitor: Known pathways regulated by GSK3 in hematopoietic stem cells are mTOR and Wnt/beta Catenin. Inhibition of Wnt/beta Catenin can release a differentiation block. Consistent with GCS-100 promoting cell differentiation, lentiviral shRNA reduced LGALS3 protein 〉 90% in THP-1 cells and increased CD11b expression, suggesting increased differentiation, compared to cells with control shRNA. GCS-100 was tested in an in vitro model of the bone marrow microenvironment using BM-derived mesenchymal stromal cell (MSC). MSC can protect leukemia cells from a variety of clinically relevant chemotherapy drugs including AraC. GCS-100 was effective at killing AML cells despite the presence of MSC. Both THP-1 and OCI-AML3 cells exhibited 〉 80% and 〉 60% reduction of viable cells, respectively, despite the presence of MSC when treated with 250 ug/mL GCS-100 for 72 hours. In addition, GCS-100 was found to block adhesion of OCI-AML3 cells to MSC suggesting that GCS-100 could be effective in mobilizing AML cells. In summary, our findings suggest that GCS-100 can induce apoptosis in AML cells as a single agent or in combination with the BH3 mimetic ABT-737. The agent is effective even in the presence of MSC suggesting it could be efficacious in the leukemia niche. These findings suggest GCS-100 could be effective for AML therapy. Disclosures Rolke: La Jolla Pharmaceutical Company: Employment. Tidmarsh:La Jolla Pharmaceutical Company: Employment.