Molecular Mechanisms of Quizartinib Response in FLT3-ITD Negative AML: An Updated Analysis from the Pethema Quiwi Trial
Authors: Mosquera Orgueira Adrian , Perez Encinas Manuel , Pérez Míguez Carlos , Rodriguez Veiga Rebeca , Bergua Burgues Juan Miguel , Lorenzo Algarra Jesus , Botella Carmen , Perez Simon Jose Antonio , Bernal Teresa , Calabuig Marisa , Calbacho Maria , Salamero Olga , Serrano Josefina , Noriega Victor , Lopez Lopez Juan Antonio , Vives Susana , Colorado Mercedes , Lopez Lorenzo Jose Luis , Vidriales Vicente Maria , Garcia Boyero Raimundo , Olave Rubio Maria Teresa , Herrera Puente Pilar , Arce Olga , Barrios Garcia Manuel , Sayas Lloris Maria Jose , Polo Marta , Gomez Roncero Maria Isabel , Barragán Eva , Ayala Rosa , Chillon Carmen , Calasanz Maria Jose , Boluda Blanca , Peleteiro Raindo Andres , Amigo Raquel , Martinez-Cuadron David , Labrador Jorge , Montesinos Pau 
Published: December 2024

Introduction
FLT3 ITD negative acute myeloid leukemia (AML) remains challenging to treat due to limited targeted therapeutic options. The QUIWI trial showed promising outcomes with quizartinib (Quiza) combined with standard chemotherapy in this setting. This analysis aims to compare transcriptional profiles of long-term survivors to hypothesize molecular mechanisms underlying differential responses to Quiza versus placebo.
Methods
RNA sequencing (RNAseq) was performed on an expanded cohort of 243 patients from the QUIWI trial, with RNA extracted, assessed for integrity (TapeStation), and quantified (Qubit). PolyA RNAseq using TruSeq technology was conducted, followed by sequence alignment to the GRCh37 reference genome using Hisat and differential expression analysis with DESeq2. P-values were adjusted using the FDR method. Gene ontology and pathway analyses were performed using the WebGestalt portal.
Results
At the time of analysis, the median follow-up was 3.25 years, and the median overall survival (OS) was not reached for the Quiza subgroup. The analysis focused on patients who survived a minimum of 24 months in both placebo and Quiza arms, identifying 267 differentially expressed genes (DEGs) with a false discovery rate (FDR) of 10% between both groups. Among the DEGs, 211 genes were overexpressed in the Quiza group, whereas 56 genes were overexpressed in the placebo group. A significant enrichment of significantly underexpressed heat shock protein genes (HSPA1A, HSPA2, DNAJA3, AHSA1, DNAJB1, FDR &lt; 5%) was observed among longer responders to Quiza. This results aligned with previous evidence indicating mechanistic relationships between FLT3 inhibitors and heat shock proteins reported by Katayama et al. (2018) and Fleischmann et al. (2021). Additionally, genes involved in nucleic acid metabolism, DNA replication, transcription, and splicing (DNTT, FTSJ3, METTL13, TWNK, SF3B3) were significantly underexpressed among longer Quiza responders compared to the placebo group. Conversely, the Quiza group exhibited a notable and significant enrichment in genes associated with the enzyme-linked receptor protein signaling pathway (FDR &lt; 5%). This included several transmembrane receptor proteins with tyrosine kinase function (FGFR3, EPHB2, AXL, ROR2), 2 protein phosphatases (PTPRU and PPM1L), and the MAPK pathway inhibitor SPRY4.
Conclusion
This analysis provides new insights into the molecular mechanisms of Quiza action in FLT3-ITD negative AML. The significant underexpression of heat shock protein genes among longer responders to Quiza suggests a potential role of these proteins as drivers of resistance. Additionally, the broader activation of tyrosine kinase genes indicates possible off-target effects contributing to the efficacy of Quiza in this subset of AML patients. These hypotheses warrant further mechanistic exploration in future studies to enhance therapeutic strategies and clinical outcomes for this challenging AML population.