Potent Ikaros Degradation By the Cereblon E3 Ligase Modulator CC-92480 Is Effective in Combination with Menin-MLL1 Inhibition in MLL1-Rearranged and NPM1-Mutant AML

Acute myeloid leukemia (AML) has a poor prognosis despite intensive therapy. Novel therapies directed at molecular drivers of AML are needed. Ongoing clinical trials with inhibitors of the Menin and Mixed Lineage Leukemia 1 (KMT2A/MLL1) protein-protein interaction for patients with MLL1-rearranged (MLL1-r) and Nucleophosmin mutant (NPM1c) AML have promising early results. We have identified Ikaros degradation as a synergistic therapeutic target with Menin-MLL1 inhibition in MLL1-r AML and identified the novel cereblon E3 ligase modulator (CELMoD) CC-92480 as an efficacious compound in vitro and in vivo in MLL1-r and NPM1c AML models.

To find genetic targets that synergize with Menin-MLL1 inhibition, we performed a genome-scale CRISPR-Cas9 functional genetic screen in the Molm-13 (MLL1-AF9) AML cell line. Using the MAGeCKFLUTE pipeline, comparison of 14 days of treatment with VTP-50469 (Menin-MLL1 inhibitor) versus DMSO identified IKZF1 deletion as one of the most negatively selected genes when treated with VTP-50469 (IKZF1 codes for the hematopoietic transcription factor Ikaros). To validate this finding, CRISPR-Cas9-based competition assays were performed in Cas9-expressing MLL1-r and NPM1c AML cell lines by monitoring of sgRNA-RFP expression over time via flow cytometry. IKZF1 was a dependency in all four MLL1-r cell lines and both NPM1c cell lines evaluated. The sgRNAs targeting IKZF1 depleted faster when cells were treated with VTP-50469.

To evaluate the effect of Ikaros protein degradation in these AML cell lines, we assessed proliferation upon treatment with three Ikaros-degrading compounds: the Immunomodulatory Imide Drug (IMiD) lenalidomide and the CELMoDs CC-220 and CC-92480. CELMoDs are a derivative class of IMiDs with much more potent Ikaros degradation (lenalidomide < CC-220 < CC-92480) and Phase I activity in multiple myeloma patients refractory to prior IMiD therapy. Cells were plated with 5 different doses per drug. Every 3 days cells were split, drug was replenished, and live cell counts were obtained using viability staining and flow cytometry. Evaluation of the absolute IC ₅₀ in 7 MLL1-r or NPM1c AML cell lines at day 9 showed that 6 cell lines were sensitive to CC-92480 (absolute IC ₅₀ range: <0.1-to-3.6 nM), 5 were sensitive to CC-220 (absolute IC ₅₀ range: 15.7-to-215.3 nM), and only two were sensitive to lenalidomide (absolute IC ₅₀ of 267.0 nM and 555.3 nM). These data suggest that greater Ikaros degradation leads to greater efficacy in vitro. Lenalidomide, CC-220, and CC-92480 all synergized with VTP-50469 to inhibit proliferation.

Apoptosis assays were performed in the MLL1-r AML cell lines Molm-13 and MV4;11 using annexin V staining following 6 days of treatment with DMSO, lenalidomide, CC-220, and CC-92480 with and without VTP-50469. For both cell lines, 5 µM lenalidomide, 1 µM CC-220, and 3 nM CC-92480 increased apoptosis less than two-fold as single agents while VTP-50469 did so only 2.5-fold and 3.7-fold compared to DMSO control. Strikingly, the combination of each of these IMiD or CELMoD doses with VTP-50469 induced apoptotic markers at least 7.5-fold compared to DMSO control. This cooperativity between Ikaros protein degradation and Menin-MLL1 inhibition could be explained by their combined targeting of a HOXA/MEIS1 transcriptional program as Ikaros degradation in MLL1-r AML cell lines perturbs expression of HOXA9 target genes without altering the expression of HOXA9 itself.

We next tested CC-92480 in four patient derived xenograft (PDX) models of AML: one NPM1c and three MLL1-r models. In these experiments, NSG-S mice were engrafted with PDX samples (>7 mice per treatment cohort). Following detection of leukemia in peripheral blood, mice were randomized to receive vehicle control, lenalidomide 50 mg/kg (in two of the four PDXs), or CC-92480 10 mg/kg for 28-55 days and survival benefit was assessed. In these models, CC-92480 increased median survival compared to DMSO by 28.3% (p < 0.05), 66% (p <0.0005), 128% (p < 0.0005), and 133% (p < 0.0001). In two of these experiments CC-92480 was also compared to lenalidomide and increased survival by 23.4% (p < 0.0005) and 28.1% (p < 0.05).

In summary, the preclinical activity of CC-92480 in MLL1-r and NPM1c AML models, particularly in combination with Menin-MLL1 inhibition, supports translation of this compound or a similarly potent, Ikaros-degrading CELMoD into clinical trials for these molecular subtypes of AML.