KO-539 Shows Synergistic Activity With Several Targeted Agents in KMT2A-Rearranged and NPM1-Mutant AML Models

Partner | Cancer Centers | <b>MD Anderson</b>

Preclinical evidence suggests that pairing KO-539 with targeted agents directed against BCL-2, BET, and CDK6 could result in superior efficacy against KMT2A-rearranged and NPM1-mutated acute myeloid leukemia.

Preclinical data presented during the 2021 ASH Annual Meeting spotlighted the molecular correlates associated with the activity of KO-539 against acute myeloid leukemia (AML) and suggest that pairing the menin inhibitor with targeted agents directed against BCL-2, BET, and CDK6 could result in superior efficacy against KMT2A-rearranged and NPM1-mutated disease.1

KO-539 was found to induce growth inhibition, differentiation, and loss of viability of AML cells with KMT2A rearrangements or NPM1 mutations in a dose-dependent manner. This was linked with attenuation levels of MEIS1, FLT3, CDK6, BCL-xL, and BCL-2, as well as an upregulation in MCL1 and CD11b.

Treatment with the agent was also found to reduce mRNA expression of MLL1 target genes and to reduce their protein expressions in immune-phenotypically–characterized AML stem-progenitor cells. KO-539 also destabilized and reduced Menin protein levels, which can be restored by combining the agent with carfilzomib (Kyprolis).

Additionally, treatment with the menin inhibitor was found to result in lethal activity against the following cells: MOLM13, MOLM13, MOLM13-TP53-R175H, and MOLM13-TP53KO. It was noted that compared with control MOLM13 or MOLM13-TP53KO cells, MOLM13-TP53-R248Q cells have been shown to be relatively resistant to the agent.

“Co-treatment with KO-539 and venetoclax [Venclexta], a BET inhibitor, or a CDK6 inhibitor is synergistically lethal against AML cells with KMT2A rearrangements or NPM1 mutations,” presenting author John W. Davis, MD, FACS, of The University of Texas MD Anderson Cancer Center, said in a poster presentation on the findings. “Compared to treatment with KO-539 or venetoclax or vehicle control, co-treatment with KO-539 and venetoclax exerted superior in vivo anti-AML efficacy without any host toxicity in a PDX model of KMT2A-rearranged AML.”

KMT2A is a large transcriptional regulator comprised of 3696 amino acids, and a histone-lysine-N-methyltransferase. N-terminal 1400 amino acids of this marker act as a transcription factor that comprises Menin-binding domains (MBDs). Additionally, the C-terminal SET domain serves as a histone methyltransferase that mediates histone H3 lysine 4 trimethylation.

Menin is a 610 amino acid that is encoded by the MEN1 gene and binds to MBD within the N-terminal 1-40 residues of KMT2A; this forms a Menin-MLL-LEDGF ternary complex, which ties KMT2A to chromatin, Davis explained. The Menin-KMT2A complex plays an important role in regulating HOX genes cluster, which encompasses the leukemogenic HOXA9 and its co-factor MEIS1 in myeloid stem-progenitor cells; these cells are involved in embryonic development and hematopoiesis.

HOXA9 operates as a pioneer factor, and with the co-factor MEIS1, it recruits CEBPαand MLL3/MLL4 to reprogram the enhancer landscape, which encourages leukemogenesis. Although MLL1 knockout is lethal to embryonic development, conditional KO undermines self-renewal of hematopoietic stem cells, according to Davis.

In AML that harbors KMT2A rearrangements, the N-terminus of the MLL1 gene is fused to the C-terminus of any of over 80 fusion partners. The fusion partners are components of the SEC complex and recruit DOT1L to elicit H3K4Me3 and H3K70Me2 marks on active chromatin; this drives aberrant expression of HOXA9, MEIS1, PBX3, MEF2C, and CDK6. Davis noted that conditional KO of MEN1 works to prevent KMT2A-rearranged disease.

Regarding NPM1-mutated AML, the wild-type menin-KMT2A complex serves as the chief oncogenic regulator of HOXA9, MEIS1, and FLT3, supporting myeloid progenitor cells to self-renew. It has been shown that when KO-539 is administered, it serves to disrupt the Menin-KMT2A interaction between proteins.

Preclinical data have demonstrated that the menin inhibitor induces differentiation and loss of viability of AML cells. Moreover, when used as a single agent and dosed continuously and daily for 3 to 6 weeks, it has produced “profound anti-leukemic activity” in several PDX models that harbor either KMT2A rearrangements or NPM1 mutations, Davis added.

KO-539 is under exploration in adult patients with relapsed or refractory AML as part of the phase 1/2a KOMET-001 trial (NCT04067336). Preliminary from the trial presented during the 2020 ASH Annual Meeting showed that among 8 evaluable patients, KO-539 monotherapy had activity in 6 patients across dose levels.2 Three of these patients received the agent at a dose of 200 mg, 1 at 400 mg, 1 at 100 mg, and 1 at 50 mg. Three patients who received the agent at 200 mg achieved stable disease (n = 1, morphological leukemia-free state (n = 1), and complete response with minimal residual disease negativity (n = 1).

In December 2021, the FDA placed a partial clinical hold on KOMET-001 following a report of a patient death from a serious adverse effect that was potentially linked with differentiation syndrome, a known toxicity associated with differentiating agents in the treatment of patients with AML.3 Although patients on the study can continue to receive KO-539, no additional patients are permitted to enroll to the trial until the hold is lifted.

In the preclinical research presented during the 2021 ASH Annual Meeting, investigators sought to further illuminate biologic effects and understand the synergistic activity of KO-539 with other novel therapies, including inhibitors of BCL-2, BET proteins, and CDK6.

For one of the analyses, investigators treated MOLM13, MV4-11, OCI-AML3, and MOLM13 TP53-R248Q cells with indicated concentrations of KO-539 and/or the BET inhibitor OTX015 for the duration of 96 hours. At treatment completion, investigators stained cells with To-Pro-3 iodide and leveraged flow cytometry to determine the percentage of non-viable cells. Using the SynergyFinder V2, delta synergy scores for each combination were calculated. Results indicated that co-treatment with KO-539 and OTX015 induced synthetic lethality in NPM1-mutated or KMT2A-rearranged AML cells with or without mutated TP53 expression.

In another analysis, investigators treated patient-derived KMT2A-rearranged or NPM1-mutated AML cells that also expressed FLT3 with the menin inhibitor and/or OTX015 for 96 hours. At the end of treatment, cells were again stained, non-viable cells were determined, and synergy scores were computed. Again, co-treatment with the 2 agents elicited synergistic lethality in these cells, irrespective of FLT3 mutational status.

KO-539 was also examined in combination with venetoclax in MOLM13, MV4-11, and OCI-AML3 cells. Following 96 hours of treatment, cells were stained, non-viable cells were determined, and synergy scores were calculated. Co-treatment with KO-529 and venetoclax resulted in synergistic lethality in AML cells that expressed KMT2A rearrangements or NPM1 mutations. Co-treatment with the agents also achieved synthetic lethality in patient-derived AML cells harboring KMT2A rearrangements or NPM1 mutations, irrespective of mutant FLT3 expression.

In another analysis, investigators engrafted mice with luciferized, patient-derived MLL-AF9 and FLT3-TKD–expressing AML cells. These mice were monitored for 5 to 7 days, were imaged by the Xenogen camera, and were randomized to equivalent bioluminescent flux. Moreover, mice received treatment with vehicle, KO-539 at 75 mg/kg daily for 5 days, and/or OTX015 at a daily dose of 30 mg/kg for 5 days, or venetoclax at a daily dose of 30 mg/kg for 5 days. Treatment was administered for a total of 2 weeks. Using the camera, total bioluminescent flux was determined for each cohort.

Results indicated that co-treatment with KO-539 and OTX015 or KO-539 and venetoclax resulted in a reduction in leukemia burden to a significantly greater extent than treatment with any of the agents alone.

References

  1. Davis JA, Fiskus WC, Daver N, et al. Clinical-stage menin inhibitor KO-539 is synergistically active with multiple classes of targeted agents in KMT2A-r and NPM1-mutant AML models. Blood. 2021;138(suppl 1):3357. doi:10.1182/blood-2021-149831
  2. Wang ES, Altman JK, Pettit KM, et al. Preliminary data on a phase 1/2A first in human study of the menin-KMT2A (MLL) inhibitor KO-539 in patients with relapsed or refractory acute myeloid leukemia. Blood. 2020;136(suppl_1):7-8. doi:10.1182/blood-2020-134942
  3. Kura Oncology provides update on phase 1b study of KO-539 in acute myeloid leukemia. News release. Kura Oncology, Inc. November 24, 2021. Accessed December 15, 2021. https://bit.ly/31c5O2A