A phase 1 trial of 8-chloro-adenosine in relapsed/refractory acute myeloid leukemia: An evaluation of safety and pharmacokinetics.

BACKGROUND: This study evaluated the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of 8-chloro-adenosine (8-Cl-Ado) in patients with relapsed/refractory acute myeloid leukemia (AML). METHODS: 8-Cl-Ado was administered daily for 5 days; the starting dose was 100 mg/m2 , the highest dose tested was 800 mg/m2 . The end points were toxicity, disease response, and PK/PD measurements. RESULTS: The predominant nonhematologic toxicity was cardiac with grade ≥3 toxicity. Plasma PK in all patients suggested heterogeneity among patients, yet, some dose-dependency for the accumulation of 8-Cl-Ado. Two 8-Cl-Ado metabolites accumulated at similar levels to 8-Cl-Ado. Cellular PK in eight patients indicated accumulation of 8-Cl-ATP, which was associated with AML blast cytoreduction in peripheral blood. The authors determined the RP2D of 8-Cl-Ado to be 400 mg/m2 . CONCLUSIONS: Given the cardiac adverse events observed, patients require monitoring for arrhythmias and QT interval during infusion. Although peripheral blood cytoreduction was observed, responses were transient, suggesting combination strategies will be required.

Pirtobrutinib inhibits wild-type and mutant Bruton's tyrosine kinase-mediated signaling in chronic lymphocytic leukemia.

Pirtobrutinib (LOXO-305), a reversible inhibitor of Bruton's tyrosine kinase (BTK), was designed as an alternative strategy to treat ibrutinib-resistant disease that develops due to C481 kinase domain mutations. The clinical activity of pirtobrutinib has been demonstrated in CLL, but the mechanism of action has not been investigated. We evaluated pirtobrutinib in 4 model systems: first, MEC-1, a CLL cell line overexpressing BTKWT, BTKC481S, or BTKC481R; second, murine models driven by MEC-1 overexpressing BTKWT or BTKC481S; third, in vitro incubations of primary CLL cells; and finally, CLL patients during pirtobrutinib therapy (NCT03740529, ClinicalTrials.gov). Pirtobrutinib inhibited BTK activation as well as downstream signaling in MEC-1 isogenic cells overexpressing BTKWT, BTKC481S, or BTKC481R. In mice, overall survival was short due to aggressive disease. Pirtobrutinib treatment for 2 weeks led to reduction of spleen and liver weight in BTKWT and BTKC481S cells, respectively. In vitro incubations of CLL cells harboring wild-type or mutant BTK had inhibition of the BCR pathway with either ibrutinib or pirtobrutinib treatment. Pirtobrutinib therapy resulted in inhibition of BTK phosphorylation and downstream signaling initially in all cases irrespective of their BTK profile, but these effects started to revert in cases with other BCR pathway mutations such as PLCG2 or PLEKHG5. Levels of CCL3 and CCL4 in plasma were marginally higher in patients with mutated BTK; however, there was a bimodal distribution. Both chemokines were decreased at early time points and mimicked BCR pathway protein changes. Collectively, these results demonstrate that pirtobrutinib is an effective BTK inhibitor for CLL harboring wild-type or mutant BTK as observed by changes in CCL3 and CCL4 biomarkers and suggest that alterations in BCR pathway signaling are the mechanism for its clinical effects. Long-term evaluation is needed for BTK gatekeeper residue variation along with pathologic kinase substitution or mutations in other proteins in the BCR pathway.

The role of p53 and p21 on 8-chloro-adenosine-induced cellular response.

8-Chloro-adenosine (8-Cl-Ado) is currently in phase I clinical trial. Activation of p53 and transactivation of p21 regulate cell fate after genotoxic insult. Using HCT-116-isogenic-cell-lines, we evaluated the role of p53/p21 after 8-Cl-Ado-mediated response. Following 30 µM 8-Cl-Ado treatment, RNA synthesis was inhibited, p53 protein was stabilized, and p21 expression was activated. None of the cell types were arrested in G1/S phase, however, cells lacking p53 were blocked in G2/M. These cells had the least increase in apoptotic cells, although clonogenic survival demonstrated equal inhibition in all 4 cell types. Collectively, irrespective of p53 and p21 status, 8-Cl-Ado-induced cytotoxicity was similar.

Synergy of Venetoclax and 8-Chloro-Adenosine in AML: The Interplay of rRNA Inhibition and Fatty Acid Metabolism.

It is known that 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets leukemic stem cells (LSCs). In a phase I clinical trial with 8-Cl-Ado in patients with refractory or relapsed (R/R) AML, we observed encouraging but short-lived clinical responses, likely due to intrinsic mechanisms of LSC resistance. LSC homeostasis depends on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. We recently reported that 8-Cl-Ado and the BCL-2-selective inhibitor venetoclax (VEN) synergistically inhibit FAO and OXPHOS in LSCs, thereby suppressing acute myeloid leukemia (AML) growth in vitro and in vivo. Herein, we report that 8-Cl-Ado inhibits ribosomal RNA (rRNA) synthesis through the downregulation of transcription initiation factor TIF-IA that is associated with increasing levels of p53. Paradoxically, 8-Cl-Ado-induced p53 increased FAO and OXPHOS, thereby self-limiting the activity of 8-Cl-Ado on LSCs. Since VEN inhibits amino acid-driven OXPHOS, the addition of VEN significantly enhanced the activity of 8-Cl-Ado by counteracting the self-limiting effect of p53 on FAO and OXPHOS. Overall, our results indicate that VEN and 8-Cl-Ado can cooperate in targeting rRNA synthesis and OXPHOS and in decreasing the survival of the LSC-enriched cell population, suggesting the VEN/8-Cl-Ado regimen as a promising therapeutic approach for patients with R/R AML.

Development and characterization of prototypes for in vitro and in vivo mouse models of ibrutinib-resistant CLL.

Although ibrutinib improves the overall survival of patients with chronic lymphocytic leukemia (CLL), some patients still develop resistance, most commonly through point mutations affecting cysteine residue 481 (C481) in Bruton's tyrosine kinase (BTKC481S and BTKC481R). To enhance our understanding of the biological impact of these mutations, we established cell lines that overexpress wild-type or mutant BTK in in vitro and in vivo models that mimic ibrutinib-sensitive and -resistant CLL. MEC-1 cell lines stably overexpressing wild-type or mutant BTK were generated. All cell lines coexpressed GFP, were CD19+ and CD23+, and overexpressed BTK. Overexpression of wild-type or mutant BTK resulted in increased signaling, as evidenced by the induction of p-BTK, p-PLCγ2, and p-extracellular signal-related kinase (ERK) levels, the latter further augmented upon IgM stimulation. In all cell lines, cell cycle profiles and levels of BTK expression were similar, but the RNA sequencing and reverse-phase protein array results revealed that the molecular transcript and protein profiles were distinct. To mimic aggressive CLL, we created xenograft mouse models by transplanting the generated cell lines into Rag2-/-γc-/- mice. Spleens, livers, bone marrow, and peripheral blood were collected. All mice developed CLL-like disease with systemic involvement (engraftment efficiency, 100%). We observed splenomegaly, accumulation of leukemic cells in the spleen and liver, and macroscopically evident necrosis. CD19+ cells accumulated in the spleen, bone marrow, and peripheral blood. The overall survival duration was slightly lower in mice expressing mutant BTK. Our cell lines and murine models mimicking ibrutinib-resistant CLL will serve as powerful tools to test reversible BTK inhibitors and novel, non-BTK-targeted therapeutics.

The Right Dose: From Phase I to Clinical Practice.

To realize the full potential of promising new anticancer drugs, it is of paramount importance to administer them at the right dose. The aim of this educational article is to provide several opportunities to optimize anticancer drug dosing, focusing on oral targeted therapies. First, therapeutic drug monitoring can optimize exposure in individual patients, if the optimal concentration is known. This approach is of particular interest in regard to oral kinase inhibitors with high interindividual pharmacokinetic variability. If exposure is related to response, then therapeutic drug monitoring is potentially feasible, although the clinical utility of this approach has not yet been established. Other approaches to reduce variability include administration of more frequent, smaller doses and administration under optimal prandial conditions. However, for many drugs, the labeled dose has not been demonstrated to be the optimal dose; for such agents, the vast majority of patients may be receiving excessive doses, which results in excessive toxicity. Furthermore, administration of lower off-label doses may reduce both medical and financial toxicity. These strategies should be applied from registration studies to clinical practice, with the goal of better optimizing anticancer treatment.

Targeting the metabolic vulnerability of acute myeloid leukemia blasts with a combination of venetoclax and 8-chloro-adenosine.

BACKGROUND: BCL-2 inhibition through venetoclax (VEN) targets acute myeloid leukemia (AML) blast cells and leukemic stem cells (LSCs). Although VEN-containing regimens yield 60-70% clinical response rates, the vast majority of patients inevitably suffer disease relapse, likely because of the persistence of drug-resistant LSCs. We previously reported preclinical activity of the ribonucleoside analog 8-chloro-adenosine (8-Cl-Ado) against AML blast cells and LSCs. Moreover, our ongoing phase I clinical trial of 8-Cl-Ado in patients with refractory/relapsed AML demonstrates encouraging clinical benefit. Of note, LSCs uniquely depend on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. VEN inhibits OXPHOS in LSCs, which eventually may escape the antileukemic activity of this drug. FAO is activated in LSCs isolated from patients with relapsed AML. METHODS: Using AML cell lines and LSC-enriched blast cells from pre-treatment AML patients, we evaluated the effects of 8-Cl-Ado, VEN and the 8-Cl-Ado/VEN combination on fatty acid metabolism, glycolysis and OXPHOS using liquid scintillation counting, a Seahorse XF Analyzer and gene set enrichment analysis (GSEA). Western blotting was used to validate results from GSEA. HPLC was used to measure intracellular accumulation of 8-Cl-ATP, the cytotoxic metabolite of 8-Cl-Ado. To quantify drug synergy, we created combination index plots using CompuSyn software. The log-rank Kaplan-Meier survival test was used to compare the survival distributions of the different treatment groups in a xenograft mouse model of AML. RESULTS: We here report that VEN and 8-Cl-Ado synergistically inhibited in vitro growth of AML cells. Furthermore, immunodeficient mice engrafted with MV4-11-Luc AML cells and treated with the combination of VEN plus 8-Cl-Ado had a significantly longer survival than mice treated with either drugs alone (p ≤ 0.006). We show here that 8-Cl-Ado in the LSC-enriched population suppressed FAO by downregulating gene expression of proteins involved in this pathway and significantly inhibited the oxygen consumption rate (OCR), an indicator of OXPHOS. By combining 8-Cl-Ado with VEN, we observed complete inhibition of OCR, suggesting this drug combination cooperates in targeting OXPHOS and the metabolic homeostasis of AML cells. CONCLUSION: Taken together, the results suggest that 8-Cl-Ado enhances the antileukemic activity of VEN and that this combination represents a promising therapeutic regimen for treatment of AML.

8-chloro-adenosine activity in FLT3-ITD acute myeloid leukemia.

Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD + MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.

A pilot study of lower doses of ibrutinib in patients with chronic lymphocytic leukemia.

Ibrutinib is highly efficacious and used at 420 mg/d for treatment of chronic lymphocytic leukemia (CLL). We previously demonstrated a decline in Bruton's tyrosine kinase (BTK) protein levels in CLL cells after 1 cycle of ibrutinib, suggesting ibrutinib dose could be lowered after the first cycle without loss of biological effect. To test this postulate, a pilot study (NCT02801578) was designed to systematically reduce ibrutinib dosing within the same patient with CLL over the course of three 28-day cycles. After an initial cycle of 420 mg/d, the dose was reduced to 280 mg/d in cycle 2, and then to 140 mg/d in cycle 3. Eleven patients began study treatment, and 9 completed the 3 cycles. Plasma and intracellular pharmacokinetics (PK), BTK occupancy, and pharmacodynamic (PD) response at different doses of ibrutinib were compared. Plasma and intracellular levels of ibrutinib were dose-dependent, and even the lowest dose was sufficient to occupy, on average, more than 95% of BTK protein. In concert, BTK downstream signaling inhibition was maintained with 140 mg/d ibrutinib in cycle 3, and there were comparable reductions in total and phospho-BTK (Tyr223) protein levels across 3 cycles. Reductions of plasma chemokine CCL3 and CCL4 levels, considered to be biomarkers of ibrutinib response, were similar during the 3 cycles. These PK/PD data demonstrate that after 1 cycle of ibrutinib at the standard 420 mg/d dose, the dose can be reduced without losing biological activity. Clinical efficacy of lower doses needs to be systematically evaluated. Such dose reductions would lower drug cost, lessen untoward toxicity, and facilitate rationale-based combinations. This trial was registered at www.clinicaltrials.gov as #NCT02801578.

Phase I studies of AZD1208, a proviral integration Moloney virus kinase inhibitor in solid and haematological cancers.

BACKGROUND: Proviral integration Moloney virus (PIM) kinases (PIM1, 2 and 3) are overexpressed in several tumour types and contribute to oncogenesis. AZD1208 is a potent ATP-competitive PIM kinase inhibitor investigated in patients with recurrent or refractory acute myeloid leukaemia (AML) or advanced solid tumours. METHODS: Two dose-escalation studies were performed to evaluate the safety and tolerability, and to define the maximum tolerated dose (MTD), of AZD1208 in AML and solid tumours. Secondary objectives were to evaluate the pharmacokinetics, pharmacodynamics (PD) and preliminary efficacy of AZD1208. RESULTS: Sixty-seven patients received treatment: 32 in the AML study over a 120-900 mg dose range, and 25 in the solid tumour study over a 120-800 mg dose range. Nearly all patients (98.5%) in both studies experienced adverse events, mostly gastrointestinal (92.5%). Dose-limiting toxicities included rash, fatigue and vomiting. AZD1208 was not tolerated at 900 mg, and the protocol-defined MTD was not confirmed. AZD1208 increased CYP3A4 activity after multiple dosing, resulting in increased drug clearance. There were no clinical responses; PD analysis showed biological activity of AZD1208. CONCLUSIONS: Despite the lack of single-agent clinical efficacy with AZD1208, PIM kinase inhibition may hold potential as an anticancer treatment, perhaps in combination with other agents.

Chlorinated adenosine analogue induces AMPK and autophagy in chronic lymphocytic leukaemia cells during therapy.

8-chloro-adenosine (8-Cl-Ado) is currently in phase-I clinical trials for acute myeloid leukaemia and chronic lymphocytic leukaemia (CLL). Previously, we demonstrated that treatment with 8-Cl-Ado leads to diminished ATP levels. We hypothesized that AMP-activated protein kinase (AMPK) signalling would be initiated in these cells, leading to induction of autophagy. AMPK activation and induction of autophagy were demonstrated during preclinical incubations in CLL cells with the analogues. Importantly, we extended similar observations in CLL lymphocytes during an 8-Cl-Ado phase-I trial. In conclusion, 8-Cl-Ado treatment induces autophagy in CLL lymphocytes in vitro as well as in vivo during clinical trial.

Idelalisib Impacts Cell Growth through Inhibiting Translation-Regulatory Mechanisms in Mantle Cell Lymphoma.

PURPOSE: PI3K is a critical node in the B-cell receptor pathway, which is responsible for survival and proliferation of B-cell malignancies. Idelalisib, a PI3Kδ-isoform-specific inhibitor, has been approved to treat B-cell malignancies. Although biological activity of the drug has been evaluated, molecular mechanisms and signaling pathway disruption leading to the biological effects of idelalisib are not yet well defined. Prior laboratory reports have identified transcription and translation as the primary events for attenuation of PI3Kα isoform. We hypothesized that PI3Kδ-isoform inhibition by idelalisib should also affect gene transcription and protein translation. EXPERIMENTAL DESIGN: Using three mantle cell lymphoma cell lines and primary cells from patients, biological consequences such as apoptosis/cell-cycle analysis, as well as RNA/protein synthesis were evaluated. Proteomics analyses (RPPA and immunoblot assays) defined molecular events downstream of PI3K/AKT cassette. RESULTS: Idelalisib treatment resulted in inhibition of protein synthesis, which correlated with reduction in cell size and cell growth. A moderate loss of viability without any change in cell-cycle profile was observed. Idelalisib treatment inhibited AKT activation, an immediate downstream PI3K effector, and also reduced phosphorylation levels of downstream AKT/mTOR pathway proteins such as PRAS40. In addition, idelalisib treatment impeded activation of the MAPK pathway, and MEK, ERK and p90RSK phosphorylation levels were reduced. Reduction in AKT, PDK1, and MEK phosphorylation correlated with protein synthesis inhibition. CONCLUSIONS: Collectively, these results clarify the molecular mechanisms of actions and may provide biomarkers and targets for combination with idelalisib in B-cell malignancies. Clin Cancer Res; 23(1); 181-92. ©2016 AACR.

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

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.

Mechanism-based combinations with Pim kinase inhibitors in cancer treatments.

Proviral integration site for Moloney murine leukemia virus (Pim) kinases is a potential therapeutic target in both hematological and solid tumors, and is up-regulated in various cancer types. In certain cases, their expression levels are positively correlated with poor clinical outcome. A number of selective Pim kinase inhibitors are under development and a few are in clinical trials. Investigations of the mechanism of actions of these drugs have demonstrated that by inhibiting Pim kinases, processes such as transcription, translation, cell cycle progression, cell survival and drug resistance are affected. Pim kinases can be upregulated by multiple growth factors, cytokines, and chemokines, which also activate redundant pathways such as phosphatidylinositide 3-kinases/protein kinase B/mammalian targets of rapamycin, and mitogen-activated protein kinases. Interestingly, Pim kinases also share substrates with these parallel pathways. To overcome this challenge, Pim kinase inhibitors were tested in combination with other therapeutic agents based on their unique mechanism of actions. Based on existing literature, we identified studies where Pim kinase inhibitors were part of the combination strategies that used targeted agents or broad-spectrum chemotherapeutic drugs (including FDA-approved agents). The addition of Pim kinase inhibitors to these treatment strategies leads to additive to synergistic cytotoxic effect in cancer cells. Depending on the compound, combination results in sequential or complementary blockage or downregulation of oncogenic pathway. In summary, these studies provide evidence for developing mechanism-based combination therapies with Pim kinase inhibitors to treat cancers.

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

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.

Combination of Pim kinase inhibitor SGI-1776 and bendamustine in B-cell lymphoma.

BACKGROUND: SGI-1776 is a small-molecule Pim kinase inhibitor that primarily targets c-MYC-driven transcription and cap-dependent translation in mantle cell lymphoma (MCL) cells. Bendamustine is an alkylating chemotherapeutic agent approved for use in B-cell lymphoma that is known to induce DNA damage and initiate response to repair. MATERIALS AND METHODS: Our studies were conducted in MCL cell lines JeKo-1 and Mino, as well as primary B-cell lymphoma samples of MCL and splenic marginal zone lymphoma (SMZL), where we treated cells with SGI-1776 and bendamustine. We measured levels of cellular apoptosis, macromolecule synthesis inhibition, and DNA damage induced by drug treatments. RESULTS: Both SGI-1776 and bendamustine effectively induced apoptosis as single agents, and when used in combination, an additive effect in cell killing was observed in MCL cell lines JeKo-1 and Mino, as well as in MCL and SMZL primary cells. As expected, SGI-1776 was effective in inducing a decrease of global RNA and protein synthesis, and bendamustine significantly inhibited DNA synthesis and generated a DNA damage response. When used in combination, the effects were intensified in DNA, RNA, and protein synthesis inhibition compared with single-agent treatments. CONCLUSION: These data provide a foundation and suggest the feasibility of using Pim kinase inhibitors in combination with chemotherapeutic agents such as bendamustine in B-cell lymphoma.

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.