TAK-243 是一种选择性 UBA1 抑制剂，在急性髓系白血病 (AML) 中显示出临床前活性,Blood

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TAK-243 是一种选择性 UBA1 抑制剂，在急性髓系白血病 (AML) 中显示出临床前活性
Blood ( IF 21.0 ) Pub Date : 2017-12-07 , DOI: 10.1182/blood.v130.suppl_1.814.814
Samir H. Barghout _{1,

2} , Parasvi Patel _{1,

2} , Xiaoming Wang ₁ , G. Wei Xu ₁ , Simon Kavanagh ₁ , Marcela Gronda ₁ , Rose Hurren ₁ , Danny V. Jeyaraju ₁ , Neil MacLean ₁ , Marc Hyer ₃ , Allison Berger ₃ , Tary Traore ₃ , Michael Sintchak ₃ , Michael Milhollen ₃ , Mark D. Minden _{1,

2} , Razq Hakem _{1,

2} , Aaron D. Schimmer ₁

Affiliation

摘要简介：泛素样修饰激活酶 1 (UBA1；UAE) 是泛素化级联中的起始酶，在该级联中，蛋白质被泛素部分标记以调节其降解或功能。与正常造血细胞相比，AML 细胞系和原代 AML 细胞具有相同水平的 UBA1 蛋白，但对这种酶的需求增加。TAK-243 是一种有效的、选择性的 UBA1 抑制剂，我们确定了 AML 中药物的临床前活性、生物学效应和耐药机制。结果：TAK-243 以浓度和时间依赖性方式降低人 AML 细胞系（OCI-AML2、TEX、U937 和 NB4）的生长和活力，处理 48 小时后 IC50 范围为 15-40 nM。在原发性 AML 样本中，大多数 (n=18/21) 对 TAK-243 敏感，在 48 小时孵育时 IC 50 < 75 nM。这些样本包括具有高风险细胞遗传学、FLT3 突变的患者和对诱导化疗无效的患者。我们还比较了 TAK-243 对原代 AML 细胞 (n=6) 和正常造血细胞 (n=6) 的影响，并证明 TAK-243 优先抑制 AML 细胞的克隆形成生长，而不是正常细胞（减少 19 倍 CFU-白血病与 CFU-GM（正常），p ≤ 0.01）。使用细胞热位移测定法 (CETSA) 在完整的 AML 细胞中测量 TAK-243 与 UBA1 和相关 E1 酶的结合。在 AML 细胞系和原发性 AML 样本中，TAK-243 在与细胞死亡相关的浓度下结合 UBA1，但仅在高得多的浓度下结合其他 E1 酶 UBA3 和 UBA6。下一个，我们评估了 TAK-243 抑制 UBA1 的生物学效应。在与细胞死亡相关的浓度下，TAK-243 降低了 OCI-AML2 细胞和原发性 AML 样本中多泛素化和单泛素化蛋白的丰度。此外，TAK-243 处理增加了 PERK 磷酸化、CHOP、XBP1s 和 ATF4，它们是蛋白毒性应激和未折叠蛋白反应的标志物。TAK-243 抑制 DNA 双链断裂 (DSB) 修复，这可以通过 3 Gy 照射后 53BP1 向 DSB 的募集减少和持续的 γH2AX 病灶来证明。我们评估了 TAK-243 在 AML 小鼠模型中的临床前疗效和毒性。将 OCI-AML2 细胞皮下 (sc) 注射到 SCID 小鼠中，当可触及肿瘤时，用 TAK-243 (20 mg/kg sc 每周两次) 治疗小鼠。TAK-243 显着延缓了小鼠的肿瘤生长（T/C=0. 02) 无毒性，小鼠体重、血清化学或器官组织学无变化证明。在从上述处理过的小鼠中分离的肿瘤和器官中，TAK-243 优先降低肿瘤中单泛素化和多泛素化蛋白的水平，而不是正常组织。作为另一个模型，将来自 2 名患者的原代 AML 细胞注射到 NOD-SCID 小鼠的股骨中。注射两周后，用 TAK-243 (20 mg/kg sc，每周两次) 治疗小鼠。治疗3周后，处死小鼠，通过流式细胞术测量未注射股骨中的AML植入。TAK-243 降低了两个测试样本中的原发性 AML 肿瘤负荷，没有毒性。使用二次移植，我们证明 TAK-243 靶向白血病干细胞。为了了解对 TAK-243 的耐药机制，我们通过培养具有增加药物浓度的细胞来选择一群 TAK-243 抗性 OCI-AML2。与野生型细胞相比，持久性细胞对 TAK-243 的耐药性高出 33 倍（IC50 757 vs 23 nM），但增殖率正常，并且对硼替佐米、柔红霉素、米托蒽醌和 NEDD8 激活酶抑制剂保持同样敏感pevonedistat。使用 CETSA，我们显示 TAK-243 与 UBA1 在抗性细胞中的结合减少。我们对跨越腺苷酸化结构域的 UBA1 外显子 12-16 和 23-24 进行了测序。抗性细胞在外显子 16 中有错义突变，导致密码子 583 (Y583C) 处的酪氨酸被半胱氨酸取代。人 UBA1 中的 Y583 对应于酵母 Uba1 中的 Y551，在其 Uba1 结合位点与 TAK-243 产生良好的相互作用。所以，预计 Y583C 替代会干扰 TAK-243 与 UBA1 的结合。结论：TAK-243 是一种有效且选择性的 UBA1 抑制剂，其对 AML 细胞的活性优于正常造血细胞。获得性突变影响药物结合，可能是临床相关的耐药机制。这些数据支持在 AML 患者中进行 TAK-243 的临床试验。披露 Hyer：武田制药国际公司：就业。伯杰：武田制药国际公司：就业。Traore：武田制药国际公司：就业。Sinchak：武田制药国际公司：就业。Milhollen：武田制药国际公司：就业。Schimmer：武田制药：研究经费；Medivir：研究经费；诺华制药：酬金。

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TAK-243 Is a Selective UBA1 Inhibitor That Displays Preclinical Activity in Acute Myeloid Leukemia (AML)

Abstract Introduction: Ubiquitin-like Modifier Activating Enzyme 1 (UBA1; UAE) is the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Compared to normal hematopoietic cells, AML cell lines and primary AML cells have equal levels of UBA1 protein, but increased requirement for this enzyme. TAK-243 is a potent and selective inhibitor of UBA1 and we determined the preclinical activity, biological effects and mechanisms of resistance to the drug in AML. Results: TAK-243 reduced growth and viability of human AML cell lines (OCI-AML2, TEX, U937 and NB4) in a concentration- and time-dependent manner with IC50's ranging from 15-40 nM after treatment for 48 hours. In primary AML samples, most (n=18/21) were sensitive to TAK-243 with an IC50<75 nM at 48 hours of incubation. These samples included patients with high-risk cytogenetics, FLT3 mutations, and patients refractory to induction chemotherapy. We also compared the effects of TAK-243 on primary AML cells (n=6) and normal hematopoietic cells (n=6) and demonstrated that TAK-243 preferentially inhibited the clonogenic growth of AML cells over normal (19-fold reduction CFU-leukemia vs CFU-GM (normal), p ≤ 0.01). Binding of TAK-243 to UBA1 and related E1 enzymes was measured in intact AML cells using the cellular thermal shift assay (CETSA). In AML cell lines and primary AML samples, TAK-243 bound UBA1 at concentrations associated with cell death, but bound other E1 enzymes UBA3 and UBA6 only at much higher concentrations. Next, we evaluated the biological effects of UBA1 inhibition by TAK-243. At concentrations associated with cell death, TAK-243 decreased the abundance of poly- and mono-ubiquitylated proteins in OCI-AML2 cells and primary AML samples. In addition, TAK-243 treatment increased PERK phosphorylation, CHOP, XBP1s and ATF4 which are markers of proteotoxic stress and unfolded protein response. TAK-243 inhibited DNA double strand break (DSB) repair as evidenced by reduced recruitment of 53BP1 to DSBs and sustained γH2AX foci after 3 Gy of irradiation. We assessed the preclinical efficacy and toxicity of TAK-243 in mouse models of AML. OCI-AML2 cells were injected subcutaneously (sc) into SCID mice, and when tumors were palpable, mice were treated with TAK-243 (20 mg/kg sc twice weekly). TAK-243 significantly delayed tumor growth in mice (T/C=0.02) with no toxicity as evidenced by no changes in mouse body weight, serum chemistry, or organ histology. In tumors and organs isolated from the above treated mice, TAK-243 preferentially reduced levels of mono- and poly-ubiquitylated proteins in tumors over normal tissues. As an additional model, primary AML cells from 2 patients were injected into the femurs of NOD-SCID mice. Two weeks after injection, mice were treated with TAK-243 (20 mg/kg sc twice weekly). After 3 weeks of treatment, mice were sacrificed, and AML engraftment in the non-injected femur was measured by flow cytometry. TAK-243 reduced primary AML tumor burden in both tested samples without toxicity. Using secondary transplantations, we demonstrated that TAK-243 had targeted the leukemic stem cells. To understand mechanisms of resistance to TAK-243, we selected a population of TAK-243-resistant OCI-AML2 by culturing cells with increasing concentrations of the drug. Persisting cells were 33-fold more resistant to TAK-243 compared to wild-type cells (IC50 757 vs 23 nM), but had a normal rate of proliferation and remained equally sensitive to bortezomib, daunorubicin, mitoxantrone and the NEDD8-activating enzyme inhibitor pevonedistat. Using CETSA, we showed reduced binding of TAK-243 to UBA1 in the resistant cells. We sequenced UBA1 exons 12-16 and 23-24 that span the adenylation domain. Resistant cells had a missense mutation in exon 16 resulting in substitution of tyrosine with cysteine at codon 583 (Y583C). Y583 in human UBA1 corresponds to Y551 in yeast Uba1, which makes a favorable interaction with TAK-243 in its Uba1 binding site. Therefore, Y583C substitution is predicted to interfere with TAK-243 binding to UBA1. Conclusions: TAK-243 is a potent and selective UBA1 inhibitor that displays preferential activity towards AML cells over normal hematopoietic cells. Acquired mutations affect drug binding and may be a clinically relevant mechanism of resistance. These data support conducting a clinical trial of TAK-243 in patients with AML. Disclosures Hyer: Takeda Pharmaceuticals International Co.: Employment. Berger: Takeda Pharmaceuticals International Co.: Employment. Traore: Takeda Pharmaceuticals International Co.: Employment. Sintchak: Takeda Pharmaceuticals International Co.: Employment. Milhollen: Takeda Pharmaceuticals International Co.: Employment. Schimmer: Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.

更新日期：2017-12-07

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