Broad Activity of Apto-253 in AML and Other Hematologic Malignancies Correlates with KLF4 Expression Level

Introduction: Aberrant expression of the homeodomain transcription factor CDX2 has recently been reported in a large proportion of AML cases. One consequence of CDX2 deregulation appears to be repressed expression of the transcription factor KLF4. Repression of KLF4 was shown to be critical for CDX2-mediated tumorigenesis, and forced genetic de-repression of KLF4 led to apoptosis of AML cells. APTO-253 is a novel small molecule that induces the expression of KLF4 and is cytotoxic to AML cell lines at low-nanomolar concentrations. We evaluated the activity of APTO-253 against a broad panel of primary specimens from patients with acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). APTO-253 was tested both as a single agent and in combinations with 2 other emerging targeted therapies, the BET bromodomain inhibitor JQ1 and the FLT3 inhibitor quizartinib.

Methods: We used an ex vivo drug sensitivity assay to determine the activity of APTO-253, JQ1, and quizartinib across increasing concentrations of each agent up to 10 μM. Combinations were tested at fixed, equimolar ratios over the same concentration range. After a 3-day ex vivo culture, cell viability was assessed using a colorimetric tetrazolium-based MTS assay, and IC₅₀ values were calculated. RNA-Seq was performed on AML specimens to permit investigation of correlations of drug sensitivity with gene expression levels.

Results: We evaluated specimens from 177 patients with a variety of hematologic malignancy diagnoses (80 AML, 72 CLL, 25 MDS/MPN). The highest frequency of APTO-253 sensitivity occurred in AML, with 43/80 (54%) samples exhibiting an IC₅₀ <1 μM. At this cutoff, 25/72 (35%) CLL samples and 3/25 (12%) MDS/MPN samples were sensitive to APTO-253. The average expression of KLF4 mRNA was 2-fold lower among AML samples with an IC₅₀ <1 µM compared to those with IC₅₀ >1 µM (p=0.07). Approximately 65% (56/87) of cases tested with a combination of APTO-253 and JQ1 showed the combination IC₅₀ to be at least 2-fold lower than the IC₅₀ of either agent alone. This enhanced efficacy of APTO-253 with JQ1 was observed across all 3 hematologic malignancies tested, whereas quizartinib enhancement of APTO-253 sensitivity was confined to AML (14/38, or 37% showed reduced IC₅₀).

Conclusions: These results support the potential of KLF4 as an important and frequently dysregulated master transcription factor in AML and suggest that the KLF4 inducer APTO-253 is effective at killing tumor cells in a majority of AML samples. The data also indicate activity of APTO-253 in other hematologic malignancies, namely CLL. Expression level of KLF4 may be one component of a biomarker for prediction of APTO-253 efficacy; a more extensive global gene expression signature analysis is under way. Finally, these data have identified prominent interaction of APTO-253 with the BET bromodomain inhibitor JQ1, as well as AML-restricted interaction of APTO-253 with the FLT3 inhibitor quizartinib, suggesting these classes of drugs as potential combination partners for APTO-253.