Inhibiting PIM-1 Is Effective in Vitro and in Vivo against ALL: A Novel Mechanistic and Potentially Clinically Relevant Druggable Target.

The outcome for patients with acute lymphoblastic leukemia (ALL) has improved greatly over the past three decades. However, the prognosis remains dismal for those with relapsed or refractory ALL despite intensified therapy. Biologically targeted agents, such as signal transduction inhibitors (STIs) have shown promise in treating leukemia. We have reported that mTOR inhibitors (MTIs) such as rapamycin (rap), RAD-001, and CCI- 779 show activity in models of murine and human ALL. However, acquired resistance to STIs remains a concern. Furthermore, the presence of cytokines such as IL-7 and TSLP can promote survival, induce STAT5 phosphorylation, and reverse the inhibitory effects of MTIs in ALL cells. We hypothesize that IL-7-mediated signaling promotes ALL cell survival and potentially contributes to MTI resistance by upregulating alternative survival pathways, such as the JAK/STAT pathway. We have evaluated the effects of inhibiting PIM-1 kinase, a known downstream target of STAT5. Using the PIM-1 inhibitor SGI- 1776 (generously provided by SuperGen, Inc.), we have found that SGI-1776 profoundly inhibited proliferation in vitro, with an IC50 of approximately 1 mM for murine and 2.5 mM for human ALL cell lines. Greater than 90% inhibition was seen at concentrations of 5 and 10 mM in murine and human ALL lines, respectively. Furthermore, a combination of 1 mM SGI-1776 and 1 ng/ml rap resulted in further inhibition than either agent alone. Because PIM-1 is regulated at the transcription level, we measured changes in PIM-1 specific mRNA levels via real time PCR after 24 hour treatment with combinations of SGI-1776, rap and IL-7 (2 ng/ml). As seen in the Table, in each treatment condition SGI-1776 significantly decreased PIM-1 mRNA. As expected, IL-7 increased PIM-1 expression. Interestingly, inhibition of mTOR signaling via rap also resulted in an apparent compensatory increase in PIM-1 mRNA, which was in turn antagonized by SGI-1776. TABLE: fold change in PIM-1 mRNA by RT-PCR Untreated SGI-1776 IL7 IL7+SGI Rap Rap+SGI Rap+IL7 Rap+IL7+SGI 1 0.1 17 1 3 0.4 22 6.5 To evaluate SGI-1776 in a clinically relevant in vivo model, NOD/SCID mice xenografted with human primary ALL cells from several samples were treated with SGI-1776 alone, SGI-1776 + rap or drug vehicle. SGI-1776 (200 mg/kg/dose daily x 5 per week by gavage) alone or with rap decreased in vivo tumor proliferation over time as compared to untreated mice. At this dose of SGI-1776, the treated mice exhibited significant side effects, including weight loss, hunched appearance with scruffy coats, decreased appetite and decreased activity. Because of this toxicity, we were not able to detect a difference in survival as a result of observed decreases in ALL burden; however these toxicities were alleviated with a reduction of SGI-1776 to 100 mg/kg/dose, and survival studies at the better-tolerated dose are ongoing. These data show that, alone and in combination with rapamycin, the PIM-1 inhibitor SGI-1776 demonstrates activity in vitro and in vivo against human ALL. Together these data suggest that PIM-1 activation can act as a mechanism of cytokine mediated MTI resistance, making PIM-1 an attractive therapeutic target for ALL.