Novel pro-survival mechanisms of PIM2 in multiple myeloma

多发性骨髓瘤（MM）占所有血液恶性肿瘤的20%，仅次于非霍奇金淋巴瘤。 淋巴瘤，预计2022年将有34，470例新发病例和12，640例死亡。尽管有新的治疗方法，MM仍然 几乎所有患者都无法治愈。治疗失败的主要原因是促生存基因的上调 机制和逐渐更大的治疗抗性-因此定义这些促生存反应将 确定关键治疗靶点。丝氨酸/苏氨酸激酶的PIM家族（PIM 1、PIM 2和PIM 3）已经被发现。 在广泛的实体瘤和血液恶性肿瘤中表现为致癌和过表达， MM疾病进展时观察到的最高水平的PIM 2过表达。PIM激酶是组成型活性的， 由于mRNA和蛋白质的半衰期很短，它们的生物学活性取决于它们对蛋白质的调节。 表情重要的是，目前假设PIM激酶的生物学效应完全是由于它们的 激酶活性带着这个假设的问题，我们最近报道说，一流的PIM 2-选择性 非ATP竞争性抑制剂JP 11646（JP）对MM增殖的抑制作用显著更强， 与ATP竞争性PIM激酶抑制剂相比，其等效地抑制PIM 2激酶活性。我们 发现JP的增加的功效不是由于对激酶活性的上级抑制，而是由于 PIM 2基因和蛋白表达下调，这在ATP竞争性PIM 2激酶中没有发生 抑制剂的这些观察结果导致我们的中心假设，即PIM 2具有关键但以前未被认识到的功能。 MM中的激酶非依赖性（Ki）促生存功能-包括维持 PIM 2基因。初步研究发现，激酶死亡的PIM 2突变体保留了显着的促生存活性。 从机制上讲，我们发现PIM 2与MM细胞中的癌基因cMYC稳定相互作用， 与几个基因启动子结合-包括PIM 2本身。此外，cMYC抑制剂下调PIM 2， 表达，提示一种新的自调节环，其中PIM 2稳定cMYC结合和反式激活 的PIM 2启动子- JP，但不是ATP竞争性PIM激酶抑制剂，破坏这个循环。另外我们 发现SLC 25 A11和UCP 2--两个编码线粒体代谢物转运蛋白的基因--受 与此一致，JP处理或PIM 2敲低选择性地破坏了MM中的氧化还原酶。 线粒体呼吸，我们表明这是依赖于脂肪酸氧化（FAO）。我们打算测试一部小说 SLC 25 A11和UCP 2过表达重编程MM代谢以有利于FAO并促进 MM存活率。由于JP在急性髓性白血病和实体瘤中具有相似的作用，因此MM中PIM 2 Ki功能 也可能发生在其他cMYC驱动的癌症中。因此，PIM 2 Ki功能的抑制可能是广泛适用的治疗方法。 靶向致癌cMYC活性的方法。提出的具体目标是：1）。为了确定激酶- PIM 2支持MM存活的独立机制，2）。阐明MM中的PIM 2自动调节 3）确定PIM 2如何独立于其激酶活性调节MM代谢。 1

Multiple myeloma (MM) comprises 20% of all hematologic malignancies, second only to non-Hodgkin’s lymphoma, with 34,470 new cases and 12,640 deaths expected in 2022. Despite new therapeutics, MM remains incurable for almost all patients. The primary cause of treatment failure is upregulation of pro-survival mechanisms and progressively greater therapy resistance - thus defining these pro-survival responses will identify key therapeutic targets. The PIM family of serine/threonine kinases (PIM1, PIM2 and PIM3) have been shown to be oncogenic and overexpressed in a wide range of solid and hematological malignancies, with the highest level of PIM2 overexpression seen with MM disease progression. PIM kinases are constitutively active, and because the mRNA and protein have short half-lives their biological activity is dictated by regulation of their expression. Importantly, it is currently assumed that biological effects of PIM kinases are entirely due to their kinase activity. Bringing this assumption into question, we recently reported that the first-in-class PIM2-selective non-ATP-competitive inhibitor JP11646 (JP) caused significantly greater suppression of MM proliferation and viability compared to ATP-competitive PIM kinase inhibitors that equivalently inhibited PIM2 kinase activity. We found that JP’s increased efficacy was not due to superior inhibition of kinase activity, but rather to an unexpected downregulation of PIM2 gene and protein expression that did not occur with the ATP-competitive PIM2 kinase inhibitors. These observations led to our central hypothesis that PIM2 has critical but previously unrecognized kinase-independent (Ki) pro-survival function in MM – including maintaining high constitutive expression of the PIM2 gene. Preliminary studies have found that kinase-dead PIM2 mutants retain significant pro-survival activity. Mechanistically, we found that PIM2 stably interacts with the oncogene cMYC in MM cells and increases cMYC binding to several gene promoters - including PIM2 itself. Moreover, a cMYC inhibitor downregulated PIM2 expression, suggesting a novel auto-regulatory loop where PIM2 stabilizes cMYC binding and trans-activation of the PIM2 promoter – JP, but not ATP-competitive PIM kinase inhibitors, disrupts this loop. Additionally, we found that SLC25A11 and UCP2 – two genes coding mitochondrial metabolite transporters – are regulated by PIM2-cMYC axis in MM. Consistent with this, JP treatment or PIM2 knockdown selectively disrupted oxidative mitochondrial respiration, which we show is dependent on fatty acid oxidation (FAO). We propose to test a novel model of SLC25A11 and UCP2 overexpression reprogramming MM metabolism to favor FAO and promoting MM survival. Since JP has similar effects in acute myeloid leukemia and in solid tumors, PIM2 Ki function in MM may also occur to other cMYC-driven cancers. As such, inhibition of PIM2 Ki function may be a broadly applicable approach to target oncogenic cMYC activity. The Specific Aims proposed are: 1). To determine the kinase- independent mechanisms by which PIM2 supports MM survival, 2). To elucidate PIM2 auto-regulation in MM cells, and 3).To establish how PIM2 regulates MM metabolism independent of its kinase activity. 1