Tatiana Zavorotinskaya

Multiple myeloma (MM) is the second most common hematologic malignancy. Despite recent treatment advances, it remains incurable. Here, we report that Pim2 kinase expression is highly elevated in MM cells and demonstrate that it is required for MM cell proliferation. Functional interference of Pim2 activity either by short hairpin RNAs or by a potent and selective small-molecule inhibitor leads to significant inhibition of MM cell proliferation. Pim inhibition results in a significant decrease of mammalian target of rapamycin C1 (mTOR-C1) activity, which is critical for cell proliferation. We identify TSC2, a negative regulator of mTOR-C1, as a novel Pim2 substrate and show that Pim2 directly phosphorylates TSC2 on Ser-1798 and relieves the suppression of TSC2 on mTOR-C1. These findings support Pim2 as a promising therapeutic target for MM and define a novel Pim2-TSC2-mTOR-C1 pathway that drives MM proliferation.

PURPOSE: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies. EXPERIMENTAL DESIGN: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low Km for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context. RESULTS: LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model. CONCLUSIONS: We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies.

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.

Proviral insertion of Moloney virus (PIM) 1, 2, and 3 kinases are serine/threonine kinases that normally function in survival and proliferation of hematopoietic cells. As high expression of PIM1, 2, and 3 is frequently observed in many human malignancies, including multiple myeloma, non-Hodgkins lymphoma, and myeloid leukemias, there is interest in determining whether selective PIM inhibition can improve outcomes of these human cancers. Herein, we describe our efforts toward this goal. The structure guided optimization of a singleton high throughput screening hit in which the potency against all three PIM isoforms was increased >10,000-fold to yield compounds with pan PIM K is < 10 pM, nanomolar cellular potency, and in vivo activity in an acute myeloid leukemia Pim-dependent tumor model is described.

Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein-histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.