The insulin receptor substrate (IRS) family encompasses six cytoplasmic adaptors that regulate numerous cellular processes such as growth, metabolism, survival, and proliferation [1]. Insulin Receptor Substrate 1 (IRS1) is differentially expressed in hematological neoplasms, suggesting its participation in hematopoiesis and neoplastic transformation [2–5]. IRS1 acts as a central adaptor in signal transduction downstream of the insulin and Insulin-like growth factor 1 (IGF1R/IR) receptors, promoting activation of the phosphoinositide 3-kinase (PI3K)-Protein kinase B (AKT)-Mechanistic target of rapamycin (mTOR) signaling cascade [1,6]. Upon ligand binding, IRS1 is phosphorylated on tyrosine residues, creating docking sites for the p85 regulatory subunit of PI3K, leading to PIP3 production and the ensuing recruitment and activation of AKT. Subsequent activation of mTOR, particularly through the mTORC1 complex, stimulates protein synthesis, cell growth, metabolic reprogramming, and inhibition of apoptosis [1,6]. In hematologic malignancies, hyperactivation of this pathway, driven by IRS1 overexpression, autocrine/paracrine IGF1 stimulation, or upstream genetic alterations, contributes to enhanced proliferation, survival, and therapeutic resistance, as well as to metabolic adaptation and interactions with the bone marrow microenvironment [5,7–9]. Thus, IRS1 not only amplifies mitogenic and anti-apoptotic signals but also integrates metabolic and oncogenic cues, positioning it as a potential therapeutic target and biomarker in leukemias and other hematologic cancers. The role of IRS1 in the BCR::ABL1 signaling pathway was first demonstrated by our research group [1,3,10], however the hypothesis of the participation of IRS1 in normal hematopoiesis has not yet been elucidated.

In this scenario, using a murine Irs1 knockout model represents an interesting tool to evaluate the function of Irs1 in normal hematopoiesis. IRS knockout mice have been extensively used for insulin signaling pathway research, particularly in studies related to diabetes, obesity, and development. Irs1 knockout models were generated and first described by two independent research groups in 1994 [11,12].

B6.129S2-Irs1smla mice carry a spontaneous nonsense mutation in serine 57 (Irs1S57X), which in homozygosis produces a knockout animal for Irs1, characterized by low weight and body size. This mutation arose spontaneously in an embryonic stem (ES) cell subclone during the generation of a SerpinB2-targeted line. Following the identification of a recombination event, the mutation was segregated from the SerpinB2 locus, mapped to a 2.78-Mb proximal interval, and backcrossed onto a C57BL/6 background. The original phenotype included reduced growth, decreased survival, and smaller body size at birth, and the colony has since been maintained under controlled breeding conditions to ensure stable transmission of the Irs1S57X allele [13]. The present study aimed to compare the hematological parameters of wild-type and heterozygous mice for the Irs1S57X mutation and to describe the fetal lethality of homozygous mice.

Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc.). Differences between groups were analyzed via two-way ANOVA with Tukey’s post-test, while Chi-square analysis was used to determine if offspring ratios deviated from Mendelian expectations. All p-values were two-sided, and the significance level was set at 5 %.

Irs1-deficient mice are characterized by a reduction in growth, impaired glucose tolerance, and a decrease in glucose uptake [11,12]. In this study, the spontaneous truncated homozygosis mutation Irs1S57X, on a pure C57BL/6 genetic background, was associated with high fetal or postnatal mortality. In heterozygosis, the S57X mutation did not result in phenotypic changes. In contrast to these findings, Selman et al. described an increased lifespan in Irs1 null females compared to wild-type and heterozygotes [16]. Irs1-deficient models generated by targeted disruption do not exhibit significant fetal or postnatal lethality [11,12]. These data suggest that the elevated lethality observed in this study may not be directly related to Irs1 deficiency.

High lethality was described in a knockout model for SerpinB2, which spontaneously acquired the Irs1S57X mutation, resulting in the total loss of Irs1 protein expression and the smla phenotype [13]. Westrick et al. described a background sensitivity in the Irs1S57X model. Mice backcrossed to C57BL/6 J (at N3 and N4 generations) exhibit less than a 2 % survival rate at two weeks of age, unlike mice backcrossed to 129S1/SvImJ [13]. Genetic background has a relevant impact on phenotype alterations in the insulin receptor pathway [17,18].

In classical Mendelian inheritance, heterozygous intercrosses are expected to generate offspring in a 1:2:1 ratio. In the present study, chi-square analysis revealed a highly significant deviation from this expected distribution at birth, with a profound underrepresentation of homozygous Irs1S57X animals. These findings indicate a markedly reduced perinatal survival associated with homozygosity. Although one homozygous animal survived until nine weeks of age, its extreme underrepresentation among offspring and the significant deviation from Mendelian proportions indicate that survival is rare and likely influenced by modifying genetic or environmental factors. Therefore, while complete lethality cannot be assumed, homozygosity for Irs1S57X markedly compromises postnatal viability. Moreover, although the total number of offspring analyzed at birth was sufficient to detect statistical deviation, the number of pregnancies evaluated at specific embryonic stages was limited. Consequently, the precise developmental window responsible for lethality cannot be definitively established and warrants further investigation in larger cohorts.

An evaluation of the progeny of heterozygous pairs showed no significant implantation failure. Homozygous fetuses developed up to day of birth (E18.5), however, they were identified as aborted or died shortly after birth. Length of gestation is strain-dependent and normally lasts 19 to 21 days [15]. Based on these findings, further hematologic studies on B6.129S2-Irs1smla/J mice should focus on fetal hematopoiesis. This study was first developed to investigate the role of Irs1 in hematopoiesis, however, the absence of Irs1 knockout mice limited the expansion of hematological analysis between genotypes. The number of pregnant female mice at different gestational ages was also insufficient to perform statistical analysis to define an average number of genotypes in each stage. The results of the current study allowed the design of a new project for fetal hematopoiesis evaluation, since the Irs1 knockout fetuses develop until day E18.5.

In summary, the Irs1S57X mutation in heterozygous mice does not affect body weight or hematological parameters. In contrast, homozygosity for Irs1S57X is associated with markedly reduced perinatal survival, preventing studies of adult hematopoiesis. The findings indicate that lethality is not due to implantation failure, as homozygous fetuses develop until E18.5. These results highlight the limitations of this model for postnatal hematopoietic research. Future studies should focus on fetal hematopoiesis to elucidate the role of Irs1 in early hematopoietic development.

The study was approved by Animal Care and Use Committee of the Institution (CEUA/FMRP - Committee on Ethics in the Use of Animals at Ribeirão Preto Medical School).

The datasets generated and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Bruna Alves Fenerich: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Jaqueline Cristina Fernandes: Investigation, Formal analysis, Writing – review & editing. Cleide Lúcia Araújo Silva: Investigation, Formal analysis, Writing – review & editing. João Agostinho Machado-Neto: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Fabiola Traina: Writing – original draft, Resources, Project administration, Funding acquisition, Data curation, Supervision, Conceptualization.