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.


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.


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.


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.

Author notes


Asterisk with author names denotes non-ASH members.