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4171 Threshold Attenuation of Perk Signaling Is Sufficient to Overcome RAS-Mediated FLT3 Inhibitor Resistance

Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research, Combination therapy, Therapies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Cheryl A C Peretz, MD1, Elaine Tran2*, Bogdan Popescu, MD3*, Vanessa E. Kennedy, MD3, Tanya Kumar1*, Lisa McGary4*, Natalie Tantisira2*, Eric Wong2*, Jose M. Rivera5*, Simayijiang Xirenayi6*, Elliot Stieglitz, MD5, Catherine C. Smith, MD3 and Juwita Hübner, MD5*

1UCSF, San Francisco, CA
2UCSF, San Francisco
3Department of Medicine, Division of Hematology/Oncology, University of California San Francisco, San Francisco, CA
4University of California San Francisco, San Francisco, CA
5Department of Pediatrics, Benioff Children's Hospital, University of California San Francisco, San Francisco, CA
6University California San Francisco, San Francisco, CA


Acute myeloid leukemia (AML) with activation of FMS-related receptor tyrosine kinase 3 (FLT3) confers a poor prognosis, principally due to disease relapse. The FLT3 inhibitor (FLT3i) gilteritinib is standard of care in relapsed/refractory FLT3-mutant AML, however its efficacy is limited by drug resistance, primarily due to RAS mutations. This suggests that RAS signaling is the critical mediator of downstream FLT3 signaling, but there is no standardized treatment for patients who relapse on FLT3i due to RAS pathway activation. Efforts to overcome resistance have been limited both by understanding of the optimal RAS effectors to target as well as the potential toxicity of simultaneously targeting FLT3 and RAS.

Here, we demonstrate that while RAS-driven resistance to gilteritinib is mediated through MAPK, not PI3K, attenuation of pERK signaling is sufficient to overcome this resistance both in vitro and in vivo.

Methods/ Results

FLT3-ITD+ cells (Molm14) with transduced NRAS mutations are resensitized to gilteritinib by MEK inhibitor treatment, proving that MAPK signaling is necessary for RAS-driven resistance. Further, expression of a constitutively active MEK construct (MEKDD) in Molm14 confers resistance to gilteritinib, showing MAPK signaling is sufficient for RAS-driven resistance. Conversely, we show that activated PI3K signaling is neither necessary nor sufficient to drive resistance, as AKT inhibition cannot resensitize NRAS-mutants to gilteritinib, and an activated AKT mutant does not confer gilteritinib resistance.

We used a doxycycline-inducible system to increase expression of MEKDD, to titrate levels of ERK signaling activation in Molm14 cells. Surprisingly, we found that the relationship between ERK phosphorylation (pERK) and FLT3i resistance was non-linear and appeared to increase at a threshold of pERK (62%). In keeping with this, suppression of pERK levels in Molm14 cells with concurrent RAS mutations to levels below 70% of baseline using the MEK inhibitor (MEKi) trametinib, was sufficient to resensitize to gilteritinib in proliferation assays. A similar phenomenon was seen in apoptosis assays. Molm14 NRAS Q61K mutant cells treated with 500nM gilteritinib plus increasing doses of trametinib showed apoptosis plateaued at the relatively low dose of 5nM trametinib. Western Blot analysis showed residual pERK activation at this dose. Thus, attenuation of MAPK signaling below a threshold, rather than its ablation, can overcome FLT3i resistance.

In a patient-derived xenograft (PDX) model of AML with concurrent FLT3-ITD and NRAS Q61K, the combination of low dose MEK inhibition (PD-901 1.5mg/kg) combined with gilteritinib was equivalent to standard dose MEK inhibition (PD-901 5mg/kg) combined with gilteritinib in suppressing outgrowth of human CD45+ cells in spleen, bone marrow, and blood of NSGS mice in comparison to vehicle-treated control. Standard dose MEK inhibition in combination with gilteritinib was also noted to be intolerable in the majority of mice with only 2 of 6 mice surviving 28 days.

Given the similar activity of low and high dose MAPK inhibition in combination with gilteritinib, we hypothesized that suppression of ERK activation below a critical level is sufficient to activate a shared transcriptional program to trigger cell cycle arrest and apoptosis. To test this hypothesis, we performed RNAseq analysis of Molm14 NRAS Q61K cells treated with gilteritinib, standard dosing of trametinib, low dose trametinib, or the combination. There is a gene expression signature of MAPK expression unique to the combination of gilteritinib and trametinib that is dose independent of trametinib, which includes MAPK regulators including ETV4 and ETV5.


MAPK is the critical effector pathway of RAS-driven gilteritinib resistance, but partial attenuation of pERK signaling is sufficient to inhibit proliferation and induce apoptosis, restoring sensitivity to FLT3i. Applied clinically, this suggests feasibility of low dose MAPK inhibition in combination with gilteritinib to treat resistant FLT3-activated AML. This strategy that would improve tolerability, a major concern in clinical use of MAPK pathway inhibitors.

Disclosures: Kennedy: Astellas: Consultancy. Smith: Cellgene: Other: Clinical trial funding; Abbvie: Honoraria, Research Funding; Genentech: Honoraria; ERASCA: Research Funding; Revolution Medicines: Research Funding; Zentalis: Other: Clinical trial funding.

*signifies non-member of ASH