Systemic Kras ablation disrupts myeloid cell homeostasis in adult mice

Significance

To assess the potential adverse effects of panKRAS and panRAS inhibitors, we have used a genetically engineered mouse model (GEMM) that allows the systemic ablation of the Kras locus. Our results indicate that loss of Kras expression induces an increase in the numbers of cells of the myeloid lineage leading to myelomonocytic metaplasia. Moreover, we provide genetic evidence that adult mice do not survive in the absence of Rat Sarcoma (RAS) proteins. Our studies may help to evaluate the potential toxic consequences of these inhibitors in the clinic.

Abstract

The KRAS oncogene has been associated with many types of cancer, including pancreatic, lung, and colorectal. For decades, its gene products were thought to be undruggable. However, during the last decade, a large battery of KRAS inhibitors selective against specific mutations (KRAS^G12C and KRAS^G12D), panKRAS inhibitors active against all KRAS isoforms, or even panRAS inhibitors, capable of inhibiting the three members of the RAS family, have been developed. In mice, the Kras locus is essential for embryonic development and can sustain adult homeostasis in the absence of Hras and Nras expression. Thus, we considered of interest to interrogate the role of the Kras locus in an experimental system to generate potentially relevant information regarding the use of panKRAS or panRAS inhibitors in the clinic. Here, we report that systemic ablation of Kras expression in adult mice does not induce significant changes in overall survival, body weight, glucose levels, metabolic profile, or heart function. In contrast, flow cytometry and histopathological analyses of organs such as blood, bone marrow, and spleen showed a significant increase of the myeloid lineage leading to myelomonocytic metaplasia. In this context, replacement of the KRAS isoforms by HRAS is sufficient to maintain adult homeostasis, suggesting that the unique properties of the Kras locus are primarily due to its pattern of expression rather than to the activity of its gene products.

Ras (Rat Sarcoma) genes play a key role in many biological pathways related to cell proliferation, differentiation, and apoptosis (1). RAS genes encode four related protein paralogues Harvey Rat Sarcoma (HRAS), Neuroblastoma RAS (NRAS), and Kirsten Rat Sarcoma (KRAS 4A and KRAS 4B), being these last ones, some of the most frequently mutated proteins in human tumors including pancreatic, lung, and colorectal cancer (2).

For decades, KRAS 4A/4B oncoproteins (from now on designated as KRAS) was considered undruggable. However, during the last few years, several inhibitors either selective for the KRAS^G12C isoform, such as Sotorasib (Amgen) (3) and Adagrasib (Mirati Therapeutics, BMS) (4), have been approved by the Food and Drug Administration (FDA). Inhibitors selective for other mutations such as KRAS^G12D (5) or panKRAS inhibitors capable of blocking all KRAS isoforms, including the wild-type KRAS protein, are being actively explored in the clinic (5, 6). More recently, panRAS inhibitors active against the three RAS paralogues have also entered clinical trials (7).

Kras has unique properties among the members of the Ras family. In genetically engineered mouse models (GEMM), germline inactivation of Kras results in embryonic lethality (8, 9). In contrast, mice develop normally in the absence of HRAS and NRAS expression, either individually or in combination, indicating that KRAS expression is sufficient to sustain both embryonic development and adult homeostasis (10). However, the effect of eliminating Kras in adult mice remains to be explored. Although the consequences of gene ablation (protein elimination, irreversibility, etc.) differ from those induced by selective inhibitors, we reasoned that understanding the effects of systemically ablating Kras expression in adult mice may provide useful information to those using panKRAS or panRAS inhibitors that inhibit the wild-type protein in clinical trials. To this end, we have developed a conditional GEMM to determine the consequences of systemically ablating Kras in adult mice and to interrogate the compensatory roles of other Ras paralogues.

Results

Generation of Kras^lox/lox Mice.

The Kras^lox allele was generated by classical homologous recombination strategies using a vector that contained the first Kras coding exon flanked by loxP sites (SI Appendix, Fig. S1A). hUBC-CreERT2 alleles were added to the strain to allow the systemic ablation of Kras by exposing the mice to a tamoxifen (TAM) diet (11). Prior results in our laboratory had illustrated that the hUBC-CreERT2 alleles are often silenced due to epigenetic alterations. Hence, we inserted a second CreERT2 allele provided by the Rosa26-CreERT2 transgene (12, 13). The resulting strain, Kras^lox/lox; Rosa26-CreERT2^KI/KI; hUBC-CreERT2^+/Tg (designated from now on as Kras^lox/lox) was subsequently used to determine the consequences of systemically ablating Kras expression in adult mice. Kras^lox/lox mice exposed to the TAM diet, designated from now on as Kras^Δ/Δ, to indicate the deletion of Kras sequences. As controls, we used a Kras^+/+; Rosa26-CreERT2^KI/KI; hUBC-CreERT2^+/Tg strain (designated from now on as Kras^+/+). Both strains had a mixed genetic background of C57BL/6 and 129/Sv. Kras^lox/lox and Kras^+/+ mice were exposed to the same TAM diet to determine the potential deleterious consequences of the continuous expression of two activated CreERT2 recombinases. In addition, we also exposed Kras^lox/lox and Kras^+/+ animals without CreERT2 alleles to the TAM diet to discard potential negative effects of this diet on adult homeostasis (14).

First, we conducted a pilot experiment to determine the extent of Cre-mediated recombination of the conditional Kras^lox alleles by isolating spleen and liver tissue of young (2-mo-old) and old (12-mo-old) Kras^lox/lox and Kras^+/+ mice exposed to a TAM diet for 12 mo. Proteomic analysis of these tissues using common and specific peptide sequences for KRAS, NRAS, and HRAS proteins (SI Appendix, Table S1) revealed the complete absence of KRAS-specific peptides in spleen tissues of Kras^∆/∆ mice (SI Appendix, Fig. S1B). In the liver, we detected minimal presence of KRAS selective peptides (SI Appendix, Fig. S1B), corresponding to about 2% of the amount present in the corresponding tissue of wild time mice. In addition, a common peptide shared by the three members of the RAS family appeared to be approximately 50% lower in both tissues of Kras^∆/∆ mice compared to those of Kras^+/+ animals, likely due to the absence of KRAS expression. As expected, the specific peptides for HRAS and NRAS paralogues were present in the same proportion in spleen and liver tissues of TAM-exposed Kras^∆/∆ and Kras^+/+ mice indicating that the absence of KRAS expression in young and old adult mice is not compensated by increase expression of the other Ras paralogues (SI Appendix, Fig. S1B). Next, we examined MAPK and PI3K signaling after Kras ablation by western blot analysis in tissues isolated from pancreas, spleen, liver, kidney, lung, and intestine of young (2-mo-old) and old (12-mo-old) mice exposed to TAM for 12 mo. We did not observe any significant differences in the levels of pERK1/2 or pAKT expression between those tissues obtained from Kras^∆/∆ mice and those of control Kras^+/+ animals (SI Appendix, Fig. S1C).

Effect of Kras Ablation in Young and Old Mice.

For this study, we used a cohort of 67 Kras^lox/lox young (2-mo-old) mice including 34 males and 33 females, and 28 Kras^+/+ 2-mo-old animals, including 17 males and 11 females. Overall survival, determined at 12 mo of TAM exposure, was 62.7% (42/67) in the Kras^lox/lox cohort and 64.3% (18/28) in the control Kras^+/+ group. In the Log-rank, Mantel-Cox test, recommended for determination of survival rates, we obtain a P value of 0.4, indicating that the ablation of the Kras alleles had no significant influence in survival (SI Appendix, Fig. S2A).

To determine whether age had an effect on survival upon Kras ablation, we conducted a similar study using a cohort of 12-mo-old animals including 53 Kras^lox/lox mice (33 males and 20 females), and 24 Kras^+/+ mice (8 males and 16 females). At 6 mo of TAM diet (18-mo-old animals) survival was 64.1% (34/53) in the Kras^lox/lox cohort indicating that age had no effect on overall survival. In this case, survival of the control Kras^+/+ group was slightly higher than in the young mice cohort, 79.2% (19/24), probably a small deviation due to the limited number of animals used in this cohort. As expected, at the 12 mo time of TAM diet (24-mo-old mice) the percentage of surviving Kras^lox/lox cohort decreased (21/53, 39.6%), but there were no significant differences (P value = 0.1 in Mantel-Cox test) between those mice devoid of Kras expression and the control Kras^+/+ animals (12/24, 50%) (SI Appendix, Fig. S2B).

Systemic Ablation of Kras Expression Does Not Affect Body Composition, Glucose Levels, Metabolic Profile, or Heart Function.

Next, we measured a series of parameters to define the overall health status of the young and old Kras^lox/lox mice including body composition (weight, fat, muscle, and bone density), glucose, metabolism, and heart function. As illustrated in SI Appendix, Fig. S3A, body weight was equally affected by the TAM diet in both cohorts of mice, Kras^∆/∆ and Kras^+/+. Although mice lost weight during the initial month of exposure to TAM diet, they recovered and maintained their body weight through the rest of the experiment (15). No significant differences were observed between young and elderly mice or between male and female animals (SI Appendix, Fig. S3A).

Densitometric analysis of fat and muscle components also failed to reveal significant differences between young and old mice that lost Kras expression throughout their body, Kras^∆/∆ and the control Kras^+/+ animals (SI Appendix, Fig. S3 B and C). Regarding bone density, we observed around 25% increase in both young Kras^∆/∆ and Kras^+/+ mice upon the 12-mo-long exposure to the TAM diet. Although this increase is statistically significant when comparing the initial (2 mo of age) and final (14 mo of age) time points, it is most likely a consequence of the physiological growth of these young mice. No significant differences were observed in the old mice cohort (SI Appendix, Fig. S3D) (16).

Glucose blood levels were measured monthly in all mice. No significant differences were observed between Kras^∆/∆ and Kras^+/+ cohorts or between young and elderly animals (SI Appendix, Fig. S4A). Finally, heart function, as measured by ejection fraction and fractional shortening parameters, also failed to identify significant differences due to either loss of Kras expression, age of the animals, diet, or gender (SI Appendix, Fig. S4 B and C).

To determine the potential effects of Kras ablation on the metabolic profile of the mice, we analyzed levels of alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (AMY), calcium (CA), creatinine (CRE), globulin (GLOB), glucose (GLU), phosphorus (PHOS), potassium (K+), sodium (NA+), total bilirubin (TBIL), total protein (TP), and urea nitrogen (BUN) in young as well as in old animals (SI Appendix, Fig. S5 A and B). Similarly, to glucose blood levels, there were no significant differences between those mice that have lost Kras expression, Kras^∆/∆, and control Kras^+/+ mice. Likewise, we did not observe any significant difference between the responses of young (TAM exposure between 2 and 14 mo of age) and older (12 to 18 mo of age) animals.

Effect of Kras Ablation on Red Blood Cell (RBC) Numbers.

Both young and elderly Kras^∆/∆ male mice showed a statistically significant decrease in RBC parameters starting after 12 wk of TAM exposure (Fig. 1). No such effect was observed in female mice (Fig. 1). RBC count dropped to 6.4 × 10⁹ units/L in young Kras^∆/∆ male mice compared to 8.2 × 10⁹ units/L in the corresponding Kras^+/+ controls. In the elderly group, these parameters were 5.3 × 10⁹ units/L in Kras^∆/∆ males and 6.6 × 10⁹ units/L in male Kras^+/+ animals, a somewhat more limited decrease (Fig. 1A). Similar results were obtained when we examined hemoglobin levels (HGB). In young male Kras^∆/∆ mice were 9.1 × 10⁹ units/L compared with 13.1 × 10⁹ units/L in control Kras^+/+ males. In elderly mice, these parameters were 8.3 × 10⁹ units/L in Kras^∆/∆ and 9.8 × 10⁹ units/L in Kras^+/+ animals (Fig. 1B). Finally, the hematocrit (HCT) parameters were 30 × 10⁹ units/L in young Kras^∆/∆ mice compared to 43.1 × 10⁹ units/L in the corresponding Kras^+/+ controls. Similar reduction levels were observed in the elderly group where the Kras^∆/∆ mice displayed 27.6 × 10⁹ units/L versus 34 × 10⁹ units/L in the Kras^+/+ controls (Fig. 1C). Despite this moderate anemia in Kras^∆/∆ male mice, they did not display obvious symptoms of distress. Finally, we did not observe any significant changes in the total number of lymphocytes in Kras^∆/∆ male or female mice as compared to those of Kras^+/+ controls (Fig. 1D).

Fig. 1.

Hematological analysis of RBCs and lymphocytes in Kras^Δ/Δ mice. (Left) Young and (Right) Old mice. Total numbers of (A) RBCs, (B) HGB, (C) HCT, and (D) lymphocytes levels, at the initial (0 mo) (2-mo-old mice) and at the last (12 mo) (14-mo-old mice) time point of TAM exposure for young mice and at the initial (0 mo) (12-mo-old mice) and at the last (6 mo) (18-mo-old mice) time point of TAM exposure for old mice. Kras^+/+ male (dark red bars) young mice n = 16 and old mice n = 17, Kras^+/+ female (light red bars) young mice n = 13 and old mice n = 20, Kras^Δ/Δ male (dark blue bars) young mice n = 38 and old mice n = 35, and Kras^Δ/Δ female (light blue bars) young mice n = 43 and old mice n = 32. Error bars indicate mean ± SEM. Statistical analysis by the two-way ANOVA test. P > 0.05; (****) P ≤ 0.001.

Systemic Kras Ablation Induces an Increase in the Number of Myeloid Cells in Peripheral Blood.

Monthly hematological analysis revealed a significant increase in components of the myeloid lineage in young as well as in old Kras^∆/∆ animals during the 12 mo of TAM exposure in young mice or the 6 mo treatment in the elder cohort (Fig. 2). No such alterations were observed in control Kras^+/+ mice, thus indicating that these alterations must be a direct consequence of the systemic loss of KRAS expression. The total number and percentage of basophils, neutrophils, eosinophils, and monocytes in the peripheral blood of young and old mice were above the normal range for these cell types, alterations observed in males and females (Fig. 2 and SI Appendix, Table S2). The increase in eosinophils or monocytes was initially observed after 24 wk of TAM exposure in young mice or after 12 wk in the older animals. The increase in basophils and neutrophils could not be identified until 24 wk of TAM diet, a phenomenon observed in both young and old mice.

Fig. 2.

Increased numbers of myeloid cells in Kras^Δ/Δ mice. (Left) Young mice (2-mo-old animals at the start of the TAM treatment) and (Right) Old mice (12-mo-old mice at the start of the TAM treatment). (A–D) (Left) Total numbers and (Right) percentage of (A) basophils, (B) neutrophils, (C) eosinophils, and (D) monocytes and at the initial time point (0 mo) (2-mo-old mice) and at the end (12 mo) (14-mo-old mice) of TAM exposure. (E–H) (Left) Total numbers and (Right) percentage of (E) basophils, (F) neutrophils, (G) eosinophils, and (H) monocytes at the initial time point (0 mo) (12-mo-old mice) and at the end (6 mo) (18-mo-old mice) of TAM exposure. Kras^+/+ male (dark red bars) young mice n = 16 and old mice n = 17, Kras^+/+ female (light red bars) young mice n = 13 and old mice n = 20, Kras^Δ/Δ male (dark blue bars) young mice n = 38 and old mice n = 35 and Kras^Δ/Δ female (light blue bars) young mice n = 43 and old mice n = 32. Error bars indicate mean ± SEM. Statistical analysis was performed by the two-way ANOVA test. Nonstatistical difference: ns; P > 0.05; (****) P ≤ 0.001.

In the group of young Kras^∆/∆ male mice, the total number of basophils was around 36-fold higher than in the control Kras^+/+ male animals. In the case of female mice, the increase was a bit more moderate but still close to 21-fold higher in the Kras^∆/∆ females than in the control animals that retained Kras expression (Fig. 2A). In the young Kras^∆/∆ mice, the total number of neutrophils increased around 19-fold in males and 16-fold in females compared to the Kras^+/+ cohort (Fig. 2B). Eosinophils were 25 times more increased in male Kras^∆/∆ mice and up to around 74 times in female Kras^∆/∆ mice in comparation with control Kras^+/+ animals (Fig. 2C). In total numbers, the increase in monocytes was 10 times higher in male Kras^∆/∆ mice and around 6 in female Kras^∆/∆ animals (Fig. 2D).

In older mice, we also observed a statistically significant increase in the total number of basophils (sevenfold increase) in only Kras^∆/∆ females exposed for 6 mo to the TAM diet (Fig. 2E). The total number of neutrophils increased 2.6 times in male Kras^∆/∆ and around 5 in Kras^∆/∆ females as compared with the corresponding Kras^+/+ controls (Fig. 2F). We also observed an increase in the total number of eosinophils in this elderly group. Yet, it was only statistically significant in Kras^∆/∆ females, where the final number was fourfold than in Kras^+/+ females (Fig. 2G). Finally, the increase in the total number of monocytes was also exclusively observed in Kras^∆/∆ females which had around 3 times more at the end of the study than Kras^+/+ females (Fig. 2H).

Mice Develop Myelomonocytic Metaplasia in the Absence of Kras Expression Regardless of Age.

Tissues of young Kras^∆/∆ and Kras^+/+ mice exposed to TAM diet for 12 mo were collected and studied at the histological level (Fig. 3). First, we observed inflammation in areas of the intestine as well as in testis and uterus due to the TAM diet, since they were observed in ablated Kras^lox/lox and Kras^+/+ animals devoid of the CreERT2 alleles (17). More importantly, we observed histological changes in Kras^∆/∆ mice due to Kras ablation in the spleen, bone marrow (BM), lymph nodes, lung, and peripheral blood, which are related to the hematopoietic alterations described above. Yet, the architecture of these organs was well preserved. Finally, no additional defects were observed in the rest of the organs due to the systemic ablation of Kras expression.

Fig. 3.

Histopathological analysis of myelomonocytic metaplasia. Representative images of different stainings in paraffin-embedded sections of (A) spleen, (B) bone marrow, (C) ganglia, (D) lung, and (E) blood smear cell populations of Kras^+/+ and Kras^Δ/Δ mice. Staining included H&E, MPO, F4/80, CD45R/CD3 CD34, and May–Grünwald Giemsa. The scale bar for spleen staining represents 1 mm. The solid arrow points the white pulp in H&E staining. The scale bar for spleen Insets represents 0.2 mm. For the rest of the organs, scale bars represent 0.1 mm and for their Insets 0.05 mm.

Spleens of young Kras^∆/∆ mice exposed to the TAM diet for 12 mo displayed a decrease in their white pulp. Moreover, the lymphoid/erythroid cells seem to be replaced by myeloid cells. Immunostaining studies revealed a decrease in B lymphocytes (CD45R+) and T lymphocytes (CD3+) compensated by an increase in cells of myeloid lineage like neutrophils myeloperoxidase (MPO+) (Fig. 3A) (18). However, no significant differences in size or weight of spleen tissue could be observed between mice lacking or retaining Kras expression. Likewise, the lymphoid/erythroid cells in the BM of Kras^∆/∆ mice were also replaced by cells of myeloid lineage (Fig. 3B). However, no significant differences were observed in the ratio of lymphoid cells (CD45R+ or CD3+) and progenitor cells (CD34+) in the BM of Kras^∆/∆ and Kras^+/+ mice (Fig. 3B).

We also detected myeloid metaplasia in the lymph nodes of Kras^∆/∆ mice (Fig. 3C). In addition, the lungs of Kras^∆/∆ mice displayed some focal areas of inflammation due to the infiltration of neutrophils (MPO+), a condition expected in this type of metaplasia (Fig. 3D) (19). Finally, we observed a significant increase in myeloid cells in the peripheral blood of Kras^∆/∆ mice (Fig. 3E). Therefore, these alterations suggest that elimination of Kras expression alters the myeloid lineage, resulting in metaplasia characterized by extramedullary hematopoiesis (EMH) in spleen and other organs (20).

Flow Cytometry Analysis of Myeloid Cells in Bone Marrow and Spleen of Kras-Ablated Mice.

Next, we performed flow cytometry analysis of BM (Fig. 4) and spleen (Fig. 5) cells in both young and old mice to determine the distribution of the myeloid components at the end of the 12 and 6 mo TAM exposure, respectively. In BM of young Kras^∆/∆ mice, 66% of the total cell population corresponded to cells of the myeloid cells, whereas they only represented 38% in the Kras^+/+ control animals. Within the myeloid population, 49% of the cells were neutrophils in Kras^∆/∆ mice versus 27% in Kras^+/+ controls. The eosinophil population represented 4% of the total myeloid cells in Kras^∆/∆ animals compared to 3% in the Kras^+/+ controls. Finally, monocytes represented 5% of the total myeloid cells in Kras^∆/∆ mice versus 2% in Kras^+/+ controls, a result similar to that observed for macrophages that represented 5% of the myeloid cells in Kras^∆/∆ mice versus 3% in the Kras^+/+ controls (Fig. 4A). In the old mouse cohort, we also observed a significant increase in the number of myeloid cells representing 72% of the total in Kras^∆/∆ animals compared to 43% in the Kras^+/+ controls. Among the myeloid compartments, the neutrophil population represented 46% of the total in Kras^∆/∆ animals versus 29% in Kras^+/+ controls. Likewise, the percentage of eosinophil cells was 2% in Kras^∆/∆ compared to 1% in the Kras^+/+ group. Monocytes represented 10% in Kras^∆/∆ mice but only 3% in the Kras^+/+ controls. Finally, the macrophage population was 3% in Kras^∆/∆ versus 2% in Kras^+/+ mice (Fig. 4B).

Fig. 4.

Myeloid cell compartments in bone marrow of Kras^Δ/Δ mice as determined by flow cytometry. (A) Young and (B) Old mice by flow cytometry. Alive (Zombie ^UV−), CD11b-BUV395⁺ CD45-FITC⁺, Ly6G-APC⁺ correspond to neutrophils, Ly6G-APC⁻, CD11c-BUV737⁻, F4/80-PE⁺ correspond to eosinophils. Finally, Ly6G-APC⁻, Ly6C-BV421⁺, F4/80-PE⁻ correspond to monocytes and F4/80-PE⁺ to macrophages.

Fig. 5.

Myeloid cell compartments in the spleen of Kras^Δ/Δ mice as determined by flow cytometry. (A) Young and (B) Old mice by flow cytometry. Alive (Zombie ^UV−), CD11b-BUV395⁺ CD45-FITC⁺, Ly6G-APC⁺ correspond to neutrophils, Ly6G-APC⁻, CD11c-BUV737⁻, F4/80-PE⁺ correspond to eosinophils. Finally, Ly6G-APC⁻, Ly6C-BV421⁺, F4/80-PE⁻ correspond to monocytes and F4/80-PE⁺ to macrophages.

A similar study was carried out in spleen cells (Fig. 5). In the young mice cohort, the percentage of the myeloid population was 47% in Kras^∆/∆ animals versus the 4% in Kras^+/+ controls. Among the myeloid population, neutrophils were 26% in Kras^∆/∆ versus 2% in Kras^+/+ mice. The eosinophil population was 1% in Kras^∆/∆ and 0.5% in the Kras^+/+ group. Finally, monocytes represented 8% in Kras^∆/∆ animals and only 0.1% in the Kras^+/+ controls, whereas macrophages represented 1% of the total myeloid population in the ablated mice versus 0.5% in the control animals (Fig. 5A).

In the old mice cohorts, the results were similar showing an increase in all the main components of the myeloid cell population. The ratios of the total population of myeloid cells were proportional to that observed in young animals, 69% in Kras^∆/∆ mice versus 13% in Kras^+/+ controls. Neutrophils showed this trend representing 15% of the myeloid population in Kras^∆/∆ and only 0.1% in Kras^+/+ mice. Eosinophils were 1.5% in Kras^∆/∆ and only 0.2% in Kras^+/+. Monocytes represented 45% of the total myeloid cells in Kras^∆/∆ mice and only 6% in Kras^+/+ controls. Finally, macrophages were 3% in Kras^∆/∆ and only 1% in Kras^+/+ mice (Fig. 5B). These results correlate well with the increase we observed in the peripheral blood of Kras^∆/∆ mice.

Bone Marrow Progenitors Are Also Affected by the Absence of Kras Expression.

To test whether the effect of Kras ablation affects the hematopoietic progenitor compartment, we carried out flow cytometry studies on the BM of Kras^∆/∆ and Kras^+/+ mice that were in TAM treatment for nearly a year. In these studies, we checked the percentage of hematopoietic stem cells (HSC), long and short-term stem cells (LT-HSC and ST-HSC) (21–23). We observed a reduction LT- and ST-HSC in Kras^∆/∆ mice, suggesting that not only the composition of the matured myeloid cells is affected (Figs. 4 and 5) but also their progenitors (SI Appendix, Fig. S5C). These results point out an important role of Kras in maintaining the homeostasis of the myeloid lineage in the BM. Moreover, mice that lack expression of Kras display a massive differentiation of the remaining progenitors toward the hematopoietic precursors in the spleen and peripheral blood.

PanRAS Inhibitors Induce a Myeloid Transformation in K562 Cells.

To verify the possible translation of our results, we performed several assays in the human pluripotent hematopoietic cell line K562. This cell line has been demonstrated to differentiate into myelomonocytes when exposed to stimuli such as 12-O-tetradecanoylphorbol-13-acetate (TPA) in the culture medium (24). Other studies also documented that TPA induces cell growth arrest (25). Assays were performed to demonstrate the growth curve by treating K562 cells with TPA at 10 nM and with 1 and 10 nM, of the panRAS inhibitor Daraxonrasib. The results obtained demonstrated a significant reduction of cell proliferation with Daraxonrasib at both concentrations, analogous to the effect of TPA (Fig. 6A). Furthermore, transformation of the cells into myelomonocytic cells was confirmed by Hematoxylin-Eosin (HE) and May–Grünwald Giemsa, being this latter staining more efficient in identifying the transformed cells (Fig. 6 B and C). Treatment with Daraxonrasib at both concentrations resulted in a discernible transformation toward myeloid lineages, such as monocytes and neutrophils. Quantification of these transformed cells per field revealed that this transformation was significantly higher in 1 nM Daraxonrasib-treated cells than in TPA-treated cells (Fig. 6 D and E).

Fig. 6.

Effects of panRAS inhibitor Daraxonrasib in the K562 cell line. (A) Growth curve of the K562 cell line. Control without treatment (solid line), TPA treatment (red line), Daraxonrasib treatment at 1 nM (blue line) and at 10 nM (green line). (B and C) Comparative staining of (B) H&E and (C) May–Grünwald Giemsa of K562 cells untreated and treated for 3 d with TPA, Daraxonrasib 1 nM, or Daraxonrasib 10 nM. Solid arrows indicate transformed cells, monocytes, or neutrophils. Magnification: ×40. (D and E) Quantification of the number of monocytes or neutrophils in (D) H&E and (E) May–Grünwald Giemsa with each treatment. For monocytes: TPA (red bar), Daraxonrasib (Darax.) 1 nM (blue bar), and Daraxonrasib (Darax.) 10 nM (green bar). For neutrophils: TPA (light red bar), Daraxonrasib (Darax.) 1 nM (light blue bar) and Daraxonrasib (Darax.) 10 nM (light green bar). Error bars indicate mean ± SEM. Statistical analysis was performed by one-way ANOVA test. Nonstatistical difference: ns; P > 0.05; (****) P ≤ 0.001.

Complete Elimination of All Ras Paralogues.

Prior genetic studies have illustrated that whereas Kras expression is essential for mouse embryonic development, its paralogues, Hras and Nras are dispensable either individually or in combination (8–10). On the other hand, the limited consequences of Kras ablation in adult mice described in this study highlight the need to interrogate the role of the other Ras paralogues during adult homeostasis. To this end, we crossed Hras^–/– and Nras^–/– mice with Kras^lox/lox animals to generate the Hras^–/–; Nras^–/–; Kras^lox/lox; UBC-CreERT2 strain (designated, when they are under TAM diet, as Rasless mice). However, ablation of the Kras^lox conditional alleles in the absence of Hras and Nras expression induced their rapid deterioration leading to their death in few days (Fig. 7A).

Fig. 7.

Effects of complete elimination of all Ras paralogues. (A) Kaplan–Meier survival curve of (black line) Hras^−/−; Nras^−/−; Kras^lox/lox; UBC-CreERT2^+/Tg (n = 6) under TAM diet. (B) Comparative staining of H&E of the intestine of Hras^+/+; Nras^+/+; Kras^+/+; UBC-CreERT2^+/Tg (control) and Rasless mice after 8 d of TAM diet. The scale bar for the intestine represents 0.2 mm. (C) Immunohistochemical staining with (Top) Pan-Ras and (Bottom) Ki67 stainings in control and Rasless mice after 8 d of TAM diet. (D) Quantification of the Ki67 positive cells in the crypt in control and Rasless mice.

TAM-treated Rasless mice appear lethargic, with significantly reduced movement and activity levels. Histological examination of the intestinal tissue comparing with wild-type control mice (Hras^+/+; Nras^+/+; Kras^+/+; UBC-CreERT2) from Rasless mice euthanized at humane endpoint according to the guidelines of the International Guidelines for Biomedical Research Involving Animals of the Council for International Organizations of Medical Sciences revealed a damage to the tissue architecture with a reduction in the number of intestinal villi, which was responsible for their rapid death (Fig. 7B). By IHC we confirmed the efficient elimination of KRAS (Fig. 7C) and a threefold reduction in proliferation, with Ki67 marker, in the intestines from Rasless mice (Fig. 7C). We observed a positive correlation between those crypts that do not express RAS and were also negative for Ki67 (not-proliferating). On average, the quantified number of Ki67-positive cells was 47 in the control mice, which retain Kras expression, and 15 in the Rasless mice (Fig. 7D).

Kras is the only member of the Ras family that encodes two gene products, Kras4A and Kras4B, due to alternative splicing (26). Thus, we interrogated whether the ability of the Kras paralogue to sustain adult homeostasis by itself was due to a unique property of its gene products or a consequence of its pattern of expression. To this end, we used an allele, HrasKI, in which the Kras coding sequences were replaced by those of the Hras paralogue (27). These mice developed normally but displayed cardiovascular pathologies, possibly due to the increased expression of the HRAS protein by the four HRAS-encoding alleles present in this strain. Indeed, elimination of the endogenous Hras alleles in Hras^–/–; Nras^–/–; HrasKI mice no longer displayed these cardiovascular defects during adulthood at least for 1 y. These observations suggest that the HRAS protein is as functional as the combined KRASA/B isoforms, suggesting that the unique properties of the Kras locus are primarily due to its pattern of expression (27).

Discussion

In this study, we have used a conditional Kras^lox/lox strain to determine the phenotypic consequences of systemically ablating Kras expression in young (2-mo-old) and old (12-mo-old) mice exposed for 12 mo to TAM diet. Considering that KRAS expression can sustain adult homeostasis, at least in mice, we found surprising that systemic Kras ablation had no significant consequences not only in overall survival, but in body composition, blood glucose levels or metabolic profile. In contrast, flow cytometry and histopathological analyses of organs such as blood, bone marrow, and spleen showed a significant increase in cells of the myeloid lineage leading to myelomonocytic metaplasia.

Heart function was measured monthly and showed no changes between groups, despite the known role of Kras in the proliferation of cardiomyocytes (8). Indeed, elimination of Kras during embryonic development leads to extremely thin ventricular walls that resulted in the embryonic lethality beyond E11.5 (8). In addition, nononcogenic germline activating mutations in Kras lead to Noonan syndrome, a condition characterized by cardiac defects such as pulmonary valve stenosis, septal defects, and complex combinations of multiple anomalies (28, 29). Taking this into account, the lack of effect on cardiac function with the systemic deletion of Kras may be a consequence of the low expression levels of this protein in adult cardiomyocytes under basal conditions (30, 31).

The most characteristic phenotype observed in systemically ablated adult mice was an increase in the number of cells of the myeloid lineage. This increase was observed in all myeloid cells including basophils, neutrophils, eosinophils, and monocytes. This effect was observed in peripheral blood as well as in organs such as the BM and spleen. In contrast, we did not observe changes in the number of lymphocytes. A moderate anemia was also detected, yet there were no obvious symptoms of distress in the mice due to this defect.

Kras gene expression has been described to play a role in adult hematopoiesis by activating the MAPK pathway in the presence of several cytokines such as stem cell factor (SCF) and thrombopoietin (TPO) (18). The conditional deletion of Kras in adult BM leads to reduced TPO signaling in multipotent progenitors (MPPs), associated with a bias toward myeloid differentiation, accompanied by a decrease in B cells (18). At the same time, increased expression of KRAS in BM hematopoietic stem cells (HSC) leads to expansion of progenitors and B lymphocytes, but not to myeloid hyperproliferation (32). Interestingly, expression of wild-type and oncogenic Kras have opposite effects, as oncogenic Kras expression in HSCs induces fatal myeloproliferative diseases in mice (33).

These observations strongly correlate with our results and highlight the role of Kras in hematopoiesis. It is known that when there is an increase in myeloid cells in BM, a mechanism called emergency hematopoiesis is activated (34). This mechanism causes the excess of myeloid cells to be shed into other lymphoid tissues, such as the spleen and blood, a phenomenon observed by flow cytometry and tissue histopathology (34). In addition, in certain pathological conditions where the hematopoietic niche in the BM is compromised, blood cells are formed and activated outside the BM in a process known as EMH (20). This situation can be observed in malignancies characterized by hyperproliferation of myeloid cells, such as acute myeloid leukemia or primary myelofibrosis, where EMH can be seen mainly in the spleen and to a lesser extent in other organs such as the lungs and lymph nodes (20, 35, 36). This also correlates with our results, where an abnormal imbalance of hematopoietic cells can be observed in these three organs following Kras deletion, particularly in the white pulp of the spleen. The development of EMH in myeloproliferative disorders that affect HSCs in the BM is a response to displacement of lymphoid and erythroid cells by myeloid cells (20). We also observed a reduction in hematopoietic progenitors LT- and ST-HSC in BM of Kras^∆/∆ mice, indicating that it can affect not only mature immune cells but also the progenitor compartment. It is reported that TAM could induce transient immunosuppression of BM progenitors (37). The reduction of the BM precursors was only observed in Kras^∆/∆ animals and was due to the absence of Kras expression and not only due to the TAM treatment since they were compared to Kras^+/+ animals also under TAM diet. These results point out an important role of Kras in maintaining homeostasis of the myeloid lineage in the BM.

Myeloid cell disorders are usually incurable and can be classified into different pathologies depending on the cells involved, their occurrence, and their evolution. Most of them include chronic syndromes such as myeloproliferative neoplasms, myelodysplastic syndromes, and chronic myelomonocytic leukemia (38). Yet, patients with these myeloproliferative disorders have a higher risk of developing nonhematological malignancies and lymphomas, as well as other myeloid malignancies (39). HRAS and NRAS expression are dispensable for adult homeostasis in KRAS-expressing animals (10). However, they are essential for animals without KRAS expression. Indeed, ablation of Kras in mice knock out for Hras and Nras (Rasless mice) results in the rapid deterioration of their general health status and undergoes an apparent multiorgan failure that results in their death in few days.

We also performed assays with the pluripotent hematopoietic K562 cell line. This cell line, derived from a human erythroleukemia, has been extensively employed as a model for hematopoietic differentiation, given its capacity to transform into various lineages, including erythroid or monocytic, in response to specific environmental stimuli (40). The transition to the erythroid lineage has been described associated with the presence of 1-beta- D-arabinofuranosylcytosine in medium (41, 42). In contrast, the transformation to the myelomonocytic lineage has been associated with the presence of 12-O-tetradecanoylphorbol- 13-acetate (TPA) that also induces cell growth arrest in these cells (24, 25). To evaluate the potential effects of panRAS inhibitors on these cells, a series of assays were performed with Daraxonrasib. As demonstrated by Jiang et al. (7), this inhibitor has exhibited considerable inhibitory capacity in cell lines and mouse models against all RAS paralogues, including both wild-type and mutated forms. In cell growth curves, we observed a reduction in cell proliferation with Daraxonrasib treatment analogous to the reduction caused by TPA, and furthermore, the immunohistochemical study revealed that cells treated with this panRAS drug underwent transformation and maturation into monocytes and neutrophils with greater intensity and frequency compared to those treated with TPA. This preliminary approach provides a valuable basis for further investigation of the side effects of these inhibitors in a clinical setting. Correlation of these results with those observed using RAS inhibitors should be approached with caution. Aside from the obvious differences between species, it should be taken into consideration that genetic ablation is an irreversible process that leads to the complete disappearance of the KRAS protein. However, in the case of KRAS inhibitors, the protein is only temporarily affected depending on the dosing protocols, and its expression levels are unlikely to be affected. Therefore, a comparison between the results obtained by genetic approaches and those obtained with selective inhibitors should be carefully considered. Yet, the results described in this study may serve as a note of caution to monitor the myeloid cell distribution in patients treated with panKRAS and panRAS inhibitors.

Materials and Methods

Mice: Generation of the Kras^lox Allele.

The Kras genomic locus was isolated from a 129/sv genomic DNA library (Stratagene, La Jolla, CA). To generate the targeting vector, pMCG24, a phosphoglycerolkinase-hygromycin-STOP cassette (43) flanked by loxP sites was inserted in inverted orientation into a 5 Kb NotI-EcoRV DNA fragment at an EcoRI site located 100 bp upstream of the exon 1. A thymidine kinase (TK) cassette (43), was inserted within an EcoRV site located at the right homology arm (SI Appendix, Fig. S1A). Finally, a third loxP site was inserted 0.4 Kb 3’ of exon 1. The resulting targeting vector, pMCG24, was linearized and electroporated in R1 ES cells (44). Cells were maintained in culture with hygromycin (positive selection) and ganciclovir (negative selection) to select those clones with the correct recombination events. DNA from the hygromycin-resistant clones was isolated and digested with BamHI (left arm) and BamHI/XbaI (right arm) and analyzed by Southern blot using the indicated probes A and B (SI Appendix, Fig. S1A). Enzymatic digestion resulted in the following bands: 20 Kb for the wild-type and 6 Kb for the recombinant (probe A), or 5 Kb for the wild-type and 3.5 Kb for the recombinant (probe B). Positive clones for the desired recombination events were selected. A CMV-Cre recombinase expression vector was electroporated to delete the selection cassette. Cells were subcloned to obtain pure single-cell populations, microinjected into CD-1 mice blastocysts, and transferred into females. Mice that did not carry the CMV-Cre transgene were selected and intercrossed to obtain the Kras^lox/lox strain.

Additional Mouse Strains.

Hras^–/– and Nras^–/– (10), HrasKI (27), Rosa26-CreERT2 (13), and hUBC-CreERT2^Tg (11) strains have been previously reported. Studies were performed using female and male mice to account for possible effects due to sex differences.

Tamoxifen Exposure.

To induce the activity of the CreERT2 recombinases, mice were placed under a TAM diet (Tekland CRD Tam400/CreER) ad libitum.

Study Approval.

All mice used in this work were housed in the Animal Facility of the CNIO in specific pathogen-free conditions, in accordance with Federation of European Laboratory Animal Science Association recommendations and European Union legislation. All experiments described were approved by the Bioethics and Animal Welfare Committee of the Institute for Health Care Carlos III and the Autonomous Community of Madrid (PROEX 257/19) and performed following the guidelines of the International Guiding Principles for Biomedical Research Involving Animals by the Council for International Organizations of Medical Sciences.

Body Weight.

Body weight was measured weekly to ensure the good status of the mice.

Densitometry.

Densitometry was performed monthly by the Molecular Imaging Core Unit at CNIO. Mice were anesthetized with 4% inhaled isoflurane (Braun Vetcare) in 100% oxygen at a rate of 0.5 L/min in a disposable measuring tray. Densitometry was performed using the Lunar PIXImus densitometer (General Electric GE).

Echocardiogram.

Echocardiogram studies were performed monthly by the Molecular Imaging Core Unit at CNIO. Mice were anesthetized as described in densitometry assays in a warm induction chamber to avoid hypothermia. Ultrasound was performed at a shaved region using micro-ultrasound system Vevo 3100 (Visualsonics, Toronto, Canada) with an ultrasound transducer of 40 MHz (RMV704, Visualsonics, Toronto, Canada). The measured parameters were left ventricular internal diameter in systole (LVIDs) and in diastole (LVIDd). Left ventricular volume in systole (LVVs) and in diastole (LVVd) were calculated following the Teichholz formula: LVV (mm³) = [7/(2.4 + LVID] × LVID³. With all these, we calculate the Ejection Fraction (EF) and Fractional Shortening (FS) considered as measurements of heart function with these formulas: EF = [(LVVd-LVVs)/LVVd] × 100 and FS= [(LVIDd-LVIDs)/LVIDd] × 100.

Blood Analysis.

Blood was taken from the facial vein (in vivo), monthly, or directly from the heart (ex vivo). Facial vein blood was used for the glucose test performed with glu-test strips (Nova biomedical) and later analyzed by the StatStrip Xpress2 Glucose and Ketone Hospital Meter (Nova biomedical). With this same blood, blood tests were carried out using the LaserCell blood counter (CVM Diagnóstico Veterinario SL) to obtain a complete blood count (CBC), total number and percentage of white blood cells (neutrophils, monocytes, eosinophils, and basophils), and total number of lymphocytes and RBC markers (RBCs, HGB, and HCT levels). Blood from the heart was used to perform the same blood analysis described above and a metabolic profile performed with a diagnostic profiling rotor (Abaxis) on the VetScan VS2 analyzer. This analysis includes levels of alanine aminotransferase (ALT, U/L), albumin (ALB, g/dL), alkaline phosphatase (AMY, U/L), calcium (CA, mg/dL), creatinine (CRE, mg/dL), globulin (GLOB, g/dL), glucose (GLU, mg/dL), phosphorus (PHOS, mg/dL), potassium (K+, mmol/L), sodium (NA+, mmol/L), total bilirubin (TBIL, mg/dL), total protein (TP, g/dL), and urea nitrogen (BUN, mg/dL).

Mass Spectrometry.

Protein extraction from various organs (spleen, liver, lung and pancreas) was performed by incubating them with 3 zirconium beads and NP-40 lysis buffer, proteases inhibitors (complete Mini, Roche) and phosphatases inhibitors (Sigma). Samples were loaded into precast polyacrylamide gel (NuPAGE 4 to 12% Bis-Tris Midi gels, Invitrogen) and run in an electrophoresis chamber (BioRad). Gels were stained for 30 min with Coomassie Brillant Blue R-250 staining solution (Bio-Rad), and the corresponding area containing the RAS proteins was excised. After washing, proteins were reduced for 30 min at 45 °C, alkylated for 1 h at room temperature in the dark, and digested with trypsin for 16 h at 37 °C. Digestion was quenched by adding 0.1% trifluoroacetic acid and the resulting peptides desalted using C18 stage-tips. Mass spectrometry was performed by coupling an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific) to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). Peptides were loaded into a trap column (AcclaimTM PepMapTM 100 C18 LC Columns 5 μM, 20 mm length) for 3 min at a flow rate of 10 μL/min in 0.1% FA. Afterward, peptides were transferred to an EASY-Spray PepMap RSLC C18 column (2 μM, 75 μM × 50 cm, Thermo Fisher Scientific) at 45 °C and separated using a gradient at a 250 nL/min flow rate. Peptides were sprayed at 1.5 kV into the mass spectrometer via the EASY-Spray source at 300 °C and then isolated using a 1.6Th window and fragmented using Higher-energy Collisional Dissociation (HCD) with a normalized collision energy of 27 MS/MS spectra resolution was set to 60,000 (200 m/z). Peptide sequences selected (SI Appendix, Table S1) for the quantification of the different Ras paralogues were submitted to Prosit (45).

Flow Cytometry Analysis.

BM and spleen were collected from mice that had completed the TAM treatment. BM was extracted from the washed femur and tibia of mice. Spleen was minced and a single-cell suspension obtained after passing the samples through 40 µm cell strainers. All samples were incubated with ACK lysis buffer (Lonza) to lyse the erythrocytes for 3 min at room temperature. After washing them with cold Fluorescence-Activated Cell Sorting (FACS) buffer (1% BSA, 2 mM EDTA in PBS), Fc receptors were blocked by incubating the samples with 2 µL/10⁶ cells of anti-mouse CD16/CD32 (BD Pharmingen, Clone 2.4G2, Ref. 553142) at room temperature for 15 min. Cells were stained on ice for 45 min using a combination of these antibodies: FITC-CD45, BUV737-CD11c, PE-F4/80, BUV395-CD11b, BV786-SiglecF (BD Biosciences) BV421-Ly6C, and APC-Ly6G (BioLegend). For viability, Zombie UV (BioLegend) was used. Cells were processed using the LSRFortessa cytometer, and the data were analyzed using FlowJo 10.8.1. To check the number of the myeloid progenitors and their distribution, BM was extracted as described before and stained with CD3-biotin, CD115-PerCPCy5.5, CD45-BV570, CD150-BV605, Streptavidin-BV711, CD135/Flt3-APC, CD48-APCCy7, CD127-BV421 (BioLegend), Ter119-biotin (Bionova), B220-biotin, Cd11b-biotin, CD34-FITC (BD Biosciences), MHCII-UV737 (Invitrogen), c-kit/CD117-PE and Sca1/LyA/E-PECy7 (Fisher Scientific). For viability, we used Zombie Aqua (BioLegend). Cells were processed using FACSymphony A5 Cell analyzer, and data were analyzed using FlowJo 10.8.1. We selected the lineage (Lin) negative cells, which are the HSCs, and then with the receptor tyrosine kinase (c-kit) and stem cell antigen (Sca-1) markers, we selected the LSK⁺ (Lin⁻, Sca-1⁺, c-kit⁺) population. Within the LSK⁺ we differentiate between ST-HSC (CD150⁻, CD48⁻) and LT-HSC (CD150⁺, CD48⁻).

Cell Culture Assays.

The cell line K562 (46) was cultivated in Roswell Park Memorial Institute (RPMI, Sigma) media with 10% fetal bovine serum (FBS, Gibco) and 1× penicillin/streptomycin (Gibco). For cell growth assays, cells were seeded at 2 × 10⁵ cells/mL. The same day, they were treated with 10 nM of TPA (Sigma) and with 1 or 10 nM of Daraxonrasib (Revolution Medicines). Cells were collected at 24, 48, and 72 h following treatment, and their number was determined with an Automated Cell Counter (Thermo Fisher Scientific) with a Trypan blue exclusion test. The evaluation of cell morphology and classification was conducted by culturing cells at the same concentration and treatment as previously mentioned for 3 d. Thereafter, cells were collected, subjected to centrifugation using a cytospin, and stained.

Histopathology and Immunohistochemistry.

Samples were fixed in 10% buffered formalin (Sigma-Aldrich) and embedded in paraffin. For histopathological analysis, samples were serially sectioned in 3 µm thick and stained with HE, Myeloperoxidase (DAKO), CD3e Clone 2GV6 (Roche), CD45R/B220 Clone RA3-3B2 (BD Biosciences), F4/80 Clone BLR209K (Bethly), and CD34 Clone RAM34 (BD Biosciences), depending on the tissue type. For blood cell observation, blood smears were stained with May–Grünwald Giemsa (Sigma Aldrich). Cytospin cell preparations were fixed in pure acetone at room temperature for 10 min and then stained with HE and May–Grünwald Giemsa.

Western Blot Analysis.

Tissue protein extraction and western blotting were performed as previously described (47). Membranes were incubated with the following antibodies: AKT (9272 Cell Signaling Rabbit; [1:1,000]), pAKT (9271 Cell Signaling; [1:250]), ERK1 (554100 BD Biosciences; [1:1,000]), ERK2 (610103 BD Biosciences; [1:500]), pERK1/2 (9101 Cell Signaling; [1:500]), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (G8795 Sigma; [1:10,000]).

Statistical Analysis.

All statistical analyses were performed using GraphPad Prism (v8.4.0) software. Data were represented as mean ± SEM. Significance between two groups was calculated by unpaired Student’s t test, and significance between more than two groups was assessed by the one-way ANOVA test with Dunnett’s multiple comparison test. Significance between more than two groups with matched values over time was assessed by the two-way ANOVA test with Dunnett’s multiple comparison test. P values < 0.05 were considered statistically significant. Significant differences between groups were *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.