Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: (A ): Analysis of the AML patient’s microarray database (source: http://vizome.org/aml2/expression_strat/ showed that IRAK4 mRNA is significantly overexpressed in initial diagnosis (p<0.0001, n=16 each), relapse (p<0.001, n=16 each), residual disease (p=0.031, n=16 each), residual (p=0.034, n=16 each), FLT-3-ITD positive (p<0.0001, n=16 each), prior MDS (p=0.03, n=16 each), prior MPN (p<0.0001, n=16 each) and MDS- MPN+ve patients (p<0.0001, n=16 each) compared to healthy pooled CD34+ cells. On the other hand, (B ): IRAK1 mRNA expression was not significantly upregulated among these AML conditions than healthy pooled CD34+ cells. ( C ): IRAK4 mRNA is significantly upregulated in T(15:17), Inv(16)/t(16:16) and T(11q23)/MLL AML patients (source: Bloodspot.org ). ( D ): Kaplan-Meier analysis showed that IRAK4 mRNA overexpressor patients face early and greater proportions of deaths compared to low expressor patients (Source: GEPIA, http://gepia.cancer-pku.cn/detail.php?gene=IRAK4&clicktag=survival ). ( E ): IRAK1 mRNA is significantly upregulated in complex and T(11q23)/MLL karyotype presenting AML patients (source: Bloodspot.org ). ( F ): Kaplan-Meier analysis showed that IRAK1-mRNA overexpressor patients face early and great proportions of deaths than low expressor patients (Source: GEPIA, http://gepia.cancer-pku.cn/detail.php?gene=IRAK1&clicktag=survival ).

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: Microarray, Expressing, Biomarker Discovery

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: In silico docking of PSP-0119 and PSP-0102 with CRBN and IRAK1 and IRAK4 complex. (A): In silico docking showed how glutarimide ring of lenalidomide interacts with CRBN and how pyrazolyl-pyridinyl portion engages with IRAK4. Red arrows point to both interactions. (B): Individual CRBN and IRAK4 residues interacting with PSP-0119 atoms are shown. (C): In silico docking showed how glutarimide ring of lenalidomide interacts with CRBN and how pyrazolyl-pyridinyl portion engages with IRAK1. (D): Individual CRBN and IRAK1 residues interacting with PSP-0102 atoms are shown.

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: In Silico

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: ( A ): Hotspot kinase assay shows that despite drastic modifications on UR241-2, the kinase inhibition activity of PSP-0102 and PSP-0119 was not lost and showed IRAK4 kinase inhibition at 2.33nM. ( B ): PSP-0119 inhibited NF-kβ reporter activity induced by IL1β. ( C ): PSP-0119 pretreatment blocked the IL1β induced IRAK4 phosphorylation.

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: Kinase Assay, Inhibition, Activity Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: Repeated experiments showed PSP-0119 treatment did not degrade IRAK4 in THP-1 (FLT-3 wild-type) ( A ), FLT-3 wild-type AML patient ( B ) and normal bone marrow cells ( C ). In immunoblotting of FLT-3 normal AML patients ( C) , total ERK1/2 was used as a loading control as Actin and GAPDH showed variabilities. ( D ): PSP-0119 treatment degraded IRAK4 in MOLM-13 and MV-4-11 ( E ) FLT-3 mutant AML cells between doses 20-40nM during 24 hrs. of treatment ( D and E ). Normalized (IRAK4/GAPDH) pixel density is written in numbers in between the bands. ( F ): IRAK4/GAPDH normalized curves and calculated DC 50 of PSP-0119 against MV-4-11 and MOLM-13 are shown. ( G ): PS-0119 did not degrade IRAK1 in MOLM-13 cells. ( H ): PS-0119 strongly degraded IRAK1 in MV-4-11 cells.

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: Western Blot, Control, Mutagenesis

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: ( A-B ): Bulk-sequencing of PSP-0119 treated MOLM-13 cells have identified gene affected by IRAK4 degradation. ( C ): Volcano plot of the upregulated and downregulated gene after IRAK1/4 degradation in MOLM-13 cells treated with PSP-0119 compared to vehicle. PS-0119 treatment led to downregulation of metabolic driver ENOS-1 gene in MOM-13 cells. Analysis of AML patient’s microarray data available at R2-Genomics and Visualization platform showed that ENOS-1 overexpression predicted poor prognoses in AML patients.

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: Sequencing, Microarray, Over Expression