Targeting Kinase Inhibitor Induced Signaling Plasticity in Patients with Ph-Like ALL

项目摘要 重大科学和治疗进展改善了B细胞急性淋巴母细胞瘤患者的预后 白血病(B-ALL)在过去的40年里。在化疗中加入ABL1激酶抑制剂显著地 提高费城染色体阳性儿童和成人的无复发和总存活率 (ph+)所有亚型。这一巨大的治疗成功归功于Ph+白血病的致癌成瘾 对BCR-ABL1驱动的信号转导和ABL1激酶抑制剂诱导深刻的细胞毒性的能力。 费城类染色体(Ph-like)ALL是一种类似的高危白血病亚型，由多种非 BCR-ABL1基因损伤类似地激活致癌激酶信号。PH样B-ALL发生在15%-40%的 患有B-ALL的儿童和成人，与复发和死亡的风险特别高有关。我们确认了 CRLF2重排Ph样蛋白中JAK2/STAT5、PI3K和B细胞受体样蛋白(BCR)信号的组成性激活 ALL可被JAK1/2(Ruxolitinib)、PI3Kδ(Idelalisib)和src(达沙替尼)体外抑制而取消 并在临床前模型中进行体内试验。然而，尽管三重抑制会导致体外和 显著降低小鼠的白血病负担，我们怀疑这种治疗策略在临床上是可移植的 对于可能存在明显毒性的患者。因此，我们专注于发现由 JAK2-激酶抑制和鉴定：1)SGK1的高活性，这是一种已知的诱导抗药性的分子 PI3Kα突变乳腺癌患者中的PI3K抑制剂，II)PTPN6完全失活和下调 已知的磷酸酶下调JAK2，PI3K和BCR信号，以及iii)DNA损伤的积累 JAK2抑制作用。我们假设SGK1的激活和PTPN6的失活是必需的机制 以克服JAK2抑制剂诱导的信号抑制。引人注目的是，我们的初步数据表明 在JAK2抑制剂处理的样品中靶向激活的SGK1导致有效的体外细胞死亡。此外，我们 假设JAK2抑制剂诱导的DNA损伤是由RAG内切酶复合体介导的， 它在B细胞分化过程中被激活，形成BCR。已经证明JAK2/STAT5信号是 在B细胞分化过程中下调以诱导增殖停滞，从而防止细胞死亡 V(D)J重组过程中的细胞凋亡。我们的初步数据表明，我们可以利用这一保守的 JAK2抑制剂处理的类Ph细胞中靶向DNA损伤反应分子CHK1的机制 这可能会导致大量白血病细胞死亡，因为积累了未修复的DNA损伤。建议数 这项研究旨在揭示以前未知的耐药机制，并确定潜在的耐药机制 Ph-like B-ALL细胞的生物学最终建立临床前治疗方案，目标是预防 复发和治愈Ph样B-ALL患者。

Project Summary Major scientific and therapeutic advances have improved outcomes of patients with B-cell acute lymphoblastic leukemia (B-ALL) over the last four decades. Addition of ABL1 kinase inhibitors to chemotherapy has significantly improved relapse-free and overall survival of children and adults with the Philadelphia chromosome positive (Ph+) ALL subtype. This great treatment success has been attributed to oncogenic addiction of Ph+ leukemias to BCR-ABL1-driven signaling and the ability of ABL1 kinase inhibitors to induce profound cytotoxicity. Philadelphia chromosome-like (Ph-like) ALL is an analogous high-risk leukemia subtype driven by various non- BCR-ABL1 genetic lesions that similarly activate oncogenic kinase signaling. Ph-like B-ALL occurs in 15-40% of children and adults with B-ALL and is associated with a particularly high risk of relapse and death. We identified constitutively activated JAK2/STAT5, PI3K and B cell receptor-like (BCR) signaling in CRLF2-rearranged Ph-like ALL, which can be abrogated with JAK1/2 (ruxolitinib), PI3Kδ (idelalisib), and SRC (dasatinib) inhibition in vitro and in vivo in preclinical models. However, while the triple inhibition results in complete cell death in vitro and significant decrease of the leukemia burden of mice, we doubt that this treatment strategy is clinically translatable for patients given potential for appreciable toxicity. We thus focused on discovering vulnerabilities induced by JAK2-kinase inhibition and identified i) high activity of SGK1, a molecule known to induce drug resistance to PI3K inhibitors in PI3Kα-mutated breast cancer patients, ii) complete inactivity and downregulation of PTPN6, a phosphatase known to downregulate JAK2, PI3K, and BCR signaling, and iii) accumulation of DNA damage with JAK2 inhibition. We hypothesized that activation of SGK1 and inactivation of PTPN6 are required mechanisms to overcome JAK2 inhibitor-induced signaling inhibition. Strikingly, our preliminary data demonstrated that targeting activated SGK1 in JAK2 inhibitor treated samples results in potent in vitro cell death. Furthermore, we hypothesized that the JAK2-inhibitor induced DNA-damage is mediated by the RAG endonuclease complex, which is activated during B cell differentiation to form the BCR. It has been shown that JAK2/STAT5 signaling is downregulated in differentiating B cells to induce a proliferation arrest, which prevents cells from dying from apoptosis during V(D)J recombination. Our preliminary data demonstrate that we can exploit this conserved mechanism by targeting the DNA-damage response molecule CHK1 in JAK2 inhibitor treated Ph-like ALL cells, which may result in massive leukemic cell death due to accumulation of unrepaired DNA damage. The proposed study aims to uncover previously unknown mechanisms of drug resistance and to characterize the underlying biology of Ph-like B-ALL cells to ultimately establish preclinical treatment protocols with the goal to prevent relapse and to cure patients with Ph-like B-ALL.