FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript pre-clinical testing of the FLT3/IRAK4 inhibitor emavusertib, alone and in combination with other targeted drugs, against AML cells with known mutations. They showed that primary patient AML cells with FLT3 or NPM1 mutations are particularly sensitive to emavusertib and that sensitivity to the combination of emavusertib and ventoclax is correlated with high blast count and expression of CD117. This work indicates provides further clinical trials involving emavusertib, alone or in combination with venetoclax, are warranted, particularly in patients with FLT3 or NPM1 mutations.
Minor comments:
2. Figures 1 and 3 include no statistical analysis, and the conclusions are based on only a single Flt3 mutant cell line.
3. There is no description of the cut-off used to divide patient samples into susceptible and resistant groups (Figure 5).
Comments on the Quality of English LanguageThis manuscript is mostly well-written, with only minor grammar mistakes or awkward phrasing.
Author Response
Journal: CIMB (ISSN 1467-3045)
Manuscript ID: cimb-2913755
Type: Article
Title: FLT3 and IRAK4 inhibitor emavusertib in combination with BH3-mimetics in the treatment of acute myeloid leukemia
Authors: Katja Seipel * , Harpreet Mandhair , Ulrike Bacher , Thomas Pabst *
Section: Molecular Medicine
Special Issue: Molecular Research and Pathological Mechanism of Leukemia
Reviewer 1:
Comments and Suggestions for Authors
This manuscript preclinical testing of the FLT3/IRAK4 inhibitor emavusertib, alone and in combination with other targeted drugs, against AML cells with known mutations. They showed that primary patient AML cells with FLT3 or NPM1 mutations are particularly sensitive to emavusertib and that sensitivity to the combination of emavusertib and venetoclax is correlated with high blast count and expression of CD117. This work indicates that further clinical trials involving emavusertib, alone or in combination with venetoclax, are warranted, particularly in patients with FLT3 or NPM1 mutations.
Response: We thank the reviewer for the helpful comments.
Minor comments:
- Figures 1 and 3 include no statistical analysis, and the conclusions are based on only a single Flt3 mutant cell line.
Response: p values have been added to figures 1 and 3. Initial screening was done with one FLT3 mutant and three FLT3 wild type cell lines. Conclusions on the significance of FLT3 status, however, are based on 23 primary AML cells with 11 FLT3 mutated and 12 FLT3 wild type (lane 266). Cytotoxicity assays with primary AML cells may provide better indications for significance of biomarkers and treatment response than AML cell lines.
- There is no description of the cut-off used to divide patient samples into susceptible and resistant groups (Figure 5).
Response: Description of cut-off values in susceptible and resistant groups has been added (lane 287). The cut-off values of cell viability were set arbitrarily, in CA4948 and PU-H71 monotherapy at 90%, venetoclax at 80% and S63845 at 70%, in combination treatments at 85% (CA-PU), 70% (CA-VC) or 60% (CA-S).
Comments on the Quality of English Language
This manuscript is mostly well-written, with only minor grammar mistakes or awkward phrasing.
Submission Date 27 February 2024
Date of this review 15 Mar 2024 06:08:08
Date of revision: 20 March, 2024
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe preclinical study of Seipel et al. investigates the efficacy of the multikinase inhibitor (FLT3 and IRAK4) emavusertib in combination with BH3 protein targeting drugs (MCL-1 and BCL-2) and an HSP90 inhibitor. Specific efficacy and biomarkers of response were identified.
This translational study is sound and potentially interesting and opens a field for further investigation of emavusertib in AML models.
Some suggestions:
1. IC50 values of emavursertib in different cell lines should be added in a table
2. Combination index should be shown in a heatmap for each compound combined with emavusertib
3. Are synergistic doses attainable in vivo for venetoclax and emavusertib treatment? That should be discussed.
4. Has emavusertib also been tested in combination with other standard of care AML drugs (I. e. azacytidine, anthracyclines, aracytine) and is this of interest? That should also be discussed.
Author Response
Journal: CIMB (ISSN 1467-3045)
Manuscript ID: cimb-2913755
Type: Article
Title: FLT3 and IRAK4 inhibitor emavusertib in combination with BH3-mimetics in the treatment of acute myeloid leukemia
Authors: Katja Seipel * , Harpreet Mandhair , Ulrike Bacher , Thomas Pabst *
Section: Molecular Medicine
Special Issue: Molecular Research and Pathological Mechanism of Leukemia
Reviewer 2:
Comments and Suggestions for Authors
The preclinical study of Seipel et al. investigates the efficacy of the multikinase inhibitor (FLT3 and IRAK4) emavusertib in combination with BH3 protein targeting drugs (MCL-1 and BCL-2) and an HSP90 inhibitor. Specific efficacy and biomarkers of response were identified.
This translational study is sound and potentially interesting and opens a field for further investigation of emavusertib in AML models.
Response: We thank the reviewer for the helpful comments.
Some suggestions:
- IC50 values of emavursertib in different cell lines should be added in a table.
Response: IC50 is by definition the half-maximal inhibitory concentration. This measure can only be calculated in susceptible cell lines. MOLM-13 was the only cell line susceptible to CA4948, the IC50 has been indicated in the text.
- Combination index should be shown in a heatmap for each compound combined with emavusertib
Response: Combination index (CI) is used to determine the degree of drug interaction, and its formula is the sum of the ratio of the dose of each drug in the compound to the dose when used alone when the combination and compound produce 50% efficacy. Combination index values were calculated in the only CA4948 susceptible cell line MOLM-13. The CI values in the three CA4948 combination treatments have been indicated in the text.
- Are synergistic doses attainable in vivofor venetoclax and emavusertib treatment? That should be discussed.
Response: With respect to physiological dosage of the drugs we added the following information in the discussion section, lane 408. The in vitro IC50 concentrations of the tested inhibitors were determined to be in the range of physiologically relevant concentrations for all compounds. Venetoclax plasma concentrations of 1-3μg/ml (1.2-3.5μM) were observed in patients receiving 400 mg QD (Roberts et al., 2016). Emavusertib plasma concentrations of 1-5mg/ml (2-10mM) were observed in patients receiving 50 to 400mg BID (Parrondo et al., 2023). PU-H71 plasma concentrations of 1-5mM in patients infused with 100-400mg/m2 (Speranza et al., 2018). S63845 displayed impressive potency at low nanomolar concentrations in preclinical in vitro and in vivo models of hematological malignancies, including MM, AML, CML and c-MYC-driven Burkitt lymphoma (Kotschy et al., 2016).
- Has emavusertib also been tested in combination with other standard of care AML drugs (i.e. azacytidine, anthracyclines) and is this of interest? That should also be discussed.
Response: With respect to combination studies we added the following information in the introduction (lane 76). Emavusertib is currently evaluated in a phase 1/2 clinical trial in R/R AML as monotherapy and in combination with azacytidine or venetoclax (Garcia-Manero, 2022). New insights into the biology of IRAK4, development of IRAK4 inhibitors and synergies with established treatments such as chemotherapy and targeted inhibitors were discussed at the first symposium on IRAK4 in cancer (Garcia-Manero, 2024). (lane 76)
Submission Date 27 February 2024
Date of this review 11 Mar 2024 10:31:35
Date of revision: 20 March 2024.
Author Response File: Author Response.pdf
