IntroductionPim (provirus integration site for Moloney murine leukemia virus) family proteins are highly conserved serine/threonine kinases that have been implicated in cancer progression and the development of resistance to chemotherapeutic agents (for a review, see Shah et al 1 ). Three Pim kinases have been identified to date, Pim-1, -2, and -3, and elevated expression of Pim kinases have been detected in hematologic malignancies and in certain solid tumors. [2][3][4][5] These kinases have similar active sites and lack regulatory domains and thus are constitutively active if expressed. 6 The expression of Pim proteins is via the recruitment of the Janus kinase (JAK) after cytokine receptor activation, resulting in the induction of signal transducer and activator of transcription-driven transcription of PIM genes. 7 Pim kinases have been shown to be involved in several signaling pathways, and the targets identified to date are associated with the regulation of apoptosis, cell-cycle progression, differentiation, transcription, proliferation, and tumorigenesis (reviewed by Amaravadi and Thompson 6 ).PIM1 is a coactivator of MYC, and Pim kinase phosphorylation of histone H3 at serine 10 leads to stimulation of RNA polymerase II binding, which results in increased c-Myc-driven transcription. 8 It is approximated that PIM1 is required for the expression of 20% of total MYC target genes. Other target substrates include proapoptotic Bad protein, which is phosphorylated at multiple sites but predominantly at gatekeeper site Ser112 by all 3 Pim kinases. 9,10 Phosphorylation of Bad leads to its sequestration from the mitochondrial surface to the cytosol by 14-3-3 and subsequent release of antiapoptotic proteins Bcl-X L and Bcl-2. Given the oncogenic nature of Pim kinases, there has been increasing interest in developing Pim kinase inhibitors for the treatment of cancer.Pertaining to cancer biology, increased levels of Pim kinase proteins have been strongly implicated in cell survival and tumorigenesis. Elevated expression of Pim-1 has been demonstrated to induce genomic instability via disruptions in mitotic spindle checkpoints 11 and also functions to protect cells from apoptosis induced by glucocorticoids, 12 genotoxins, 13 or cytokine withdrawal. 14 Pim-1 also has been shown to interact in the p53 pathway via Mdm2. 15 Pim-1 is overexpressed in lymphomas, 16 acute leukemias, 17 and prostate cancer, 5 whereas increased expression of the human PIM2 proto-oncogene is observed in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphomas. 3 Pim-2 also is required to confer rapamycin resistance in hematopoietic cells, 18 and both Pim-1 and Pim-2 have been shown to be required for efficient pre-B-cell transformation by v-Abl oncogene. 19 More recently, Pim-3 was reported to be aberrantly expressed in colon cancer. 20 Taken together, these observations further support the rationale for the development of Pim inhibitors as therapeutic agents for hematologic malignancies. 21 We hypothesized that CLL cells would be responsive...

IntroductionPim (provirus integration site for Moloney murine leukemia virus) family proteins are highly conserved serine/threonine kinases that have been implicated in cancer progression and the development of resistance to chemotherapeutic agents (for a review, see Shah et al 1 ). Three Pim kinases (Pim-1, -2 and -3) have been identified, each with variant isoforms of the expressed protein because of alternate start sites. In humans, PIM1, PIM2, and PIM3 genes are located on chromosome 6p21, Xp11.23, and 22q13, respectively. 2,3 At the amino acid level, there is substantial homology between Pim-1 and Pim-2 (53%) 4 and Pim-3 (69%). 5 Pim kinases have overlapping functions and compensate for one another, and their numerous targets include regulators of transcription, translation, cell cycle, survival, and drug resistance ( Figure 1A).Pertaining to cancer biology, increased levels of Pim kinase proteins have been strongly implicated in cell survival and tumorigenesis. Pim kinases are overexpressed in both solid tumors such as colon, 6 prostate cancer 7 and hematologic malignancies including lymphomas, 8,9 chronic lymphocytic leukemia (CLL) 8,10 and acute leukemias. 11 Specifically in AML, up-regulation of Pim may be because of overexpression of HOXA9 12-14 and STAT activation, 15 which can act as transcription factors 16,17 for Pim. These oncogenic kinases appear to play critical roles in leukemogenesis, and resistance to chemotherapy 18 and radiotherapy. 19 Consistent with these reports, knockdown of Pim kinases was shown to impair the survival of resistant forms of FLT3-and BCR-ABL-transformed leukemia cells. 20 FLT3-ITD is one of the most prevalent activating mutations identified in AML (15%-30%; reviewed in Meshinchi et al 21 ), and is associated with inferior disease-free survival and increased relapse rate. 22 Allele analysis revealed that homozygous FLT3-ITD is a strong adverse prognostic factor in de novo AML in patients with normal cytogenetics. 23 One of the challenges of FLT3 inhibition therapy is the development of resistance and Pim-1 has been shown to contribute to this increased resistance. 24 Pim itself can phosphorylate FLT3 in a feedback loop ( Figure 1A), 25 and consequently Pim kinase inhibition may be an alternative strategy to target AML with FLT3 activating mutations.Given the oncogenic nature of Pim kinases, there has been increasing interest in developing Pim kinase inhibitors for the treatment of cancer. 26 SGI-1776 is an imidazo[1,2-b]pyridazine ( Figure 1B) small molecule that is inhibitory to all 3 Pim kinases: the IC 50 values are 7nM, 363nM, and 69nM for Pim-1, -2 and -3, respectively. 10 In addition to Pim, SGI-1776 also potently targets FLT3 (IC 50 ϭ 44nM). Using established AML cell lines with wild-type and mutated FLT3, AML mouse model system, and primary AML leukemic blasts with variety of FLT3 mutation status, we elucidate the mechanism of action of SGI-1776 and its cytotoxicity in AML. Methods DrugsSGI-1776 was obtained from SuperGen and was dissolved in DMSO and stored at Ϫ2...

Abstract 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.

BackgroundProviral 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.MethodsTwo 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.ResultsSixty-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.ConclusionsDespite 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.

IntroductionMantle cell lymphoma (MCL) is an aggressive lymphoma characterized by overexpression of cyclin D1 caused by t(11:14)(q13; q32). 1 Although current therapeutic approaches provide a good response rate (Ͼ 90%) and progression-free survival (ϳ 2.5 years), there is no effective cure for this disease. 2,3 Hence, identification of novel targets and their inhibition are needed in MCL.Proviral integration site for Moloney murine leukemia virus (Pim) kinases are oncoproteins that promote tumor progression. 4 To date, 3 Pim kinases have been identified. Pim-1, -2, and -3 are highly conserved serine/threonine/tyrosine kinases that are important for normal B-lymphocyte development 5 and that are overexpressed in B-cell malignancies, such as chronic lymphocytic leukemia (CLL) 6 and MCL. [7][8][9] In addition, Pim-1 and Pim-2 have been found to be highly expressed in other hematologic malignancies 10 as well as in solid tumors, such as prostate cancer. 11 c-Myc, also a Pim kinase substrate, has been observed to coexpress with Pim kinase in B-cell malignancies. 12 In addition, elevated Pim-1 expression in MCL has been reported to induce the p53 pathway and correlate with increased expression of MDM2. 13 Furthermore, Pim-1 expression is known to be highly associated with poor outcome in MCL patients. 7 These observations suggest that Pim kinases could be potential therapeutic targets in MCL.Pim kinase genes are early responders to growth factors and cytokines. 14 These kinases are highly conserved throughout evolution, yet Pim-1, -2, and -3 triple-knockout mice are viable and fertile, revealing the dispensability of these proteins in crucial physiologic developmental processes. 5 Pim kinases phosphorylate several substrates, including c-Myc and Histone H3 (H3) that drive the transcription process. 12,15 Pim-1 phosphorylation of Histone H3 at Ser10 has been reported to be a necessary event for c-Mycdriven transcription. 15 Pim-1 phosphorylates c-Myc at Ser62 to stabilize this protein. 16 Notably, both Pim-1 and Pim-2 work synergistically with c-Myc, as confirmed by double-knockout studies in mice. 5 The translation regulator eukaryotic elongation factor 4E-BP1 is also a substrate of Pim kinases. Pim kinases phosphorylate the priming sites Thr37/46, allowing for the hyperphosphorylation of 4E-BP1, including Ser65 phosphorylation by Pim-2, causing it to dissociate from eukaryotic initiation factor 4. 17,18 Dissociation of eukaryotic initiation factor 4 contributes to the activation of cap-dependent translation. 18,19 In addition, Pim-1 and Pim-2 phosphorylate Bcl-2-associated death promoter (Bad) at Ser112; this phosphorylation disrupts binding of Bad to the antiapoptotic protein B-cell lymphoma-extra large (Bcl-X L ) and allows Bad to bind scaffold protein 14-3-3 to sequester its proapoptotic function, thereby activating a cell survival pathway. 20,21 Furthermore, cell cycle proteins such as CDKN1B (or p27) and cell division cycle 25A/C are phosphorylated by Pim kinases and are involved in promoting proliferation. [22]...

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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-XL, 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 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.

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. 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, as wells as changes in cell cycle profile were measured. SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138+ cells (<10%) as assessed by Annexin-V/PI. 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, as assessed by 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 autophagy LC3b marker and translation initiation proteins (phospho p70S6K and 4E-BP1) corroborated autophagy induction. 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.