NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

Abstract

Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1β release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1β release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRAS^G12D mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers.

Keywords: CMML; IL-1 blockers; KRAS; NLRP3 blockers; NLRP3 inflammasome; RAS mutations; anakinra; inflammation; myelodysplastic syndromes; myeloproliferative neoplasms.

Graphical abstract

Figure 1

Evolution of autoinflammatory episodes and levels of IL-1β of CMML KRAS^G12D patient treated with anakinra (A) Picture of the cellulitis episode on the lower legs of the index patient. (B) Interleukin-1β release from peripheral blood (PB) mononuclear cells from healthy donors (n = 4, each dot represents an independent donor) and the index patient with a KRAS^G12D mutation before anakinra treatment (each dot represents a technical replicate). Data are represented as mean ± SEM. (C) Evolution of PB monocytes count during the evolution of the index patient. After introduction of anakinra, the patient was free of autoinflammatory episodes for 4 months, after which new episodes of autoinflammation occurred and anakinra was discontinued. Three weeks later, with constrictive pericarditis and congestive heart failure refractory to all therapeutic measures taken, associated with congestive nephropathy and cryptogenic organized pneumonia, in critical life-threatening condition, anakinra was reintroduced. Note the decrease of monocyte count in response to anakinra treatment (both the first time and when restored). (D) Left orchiepididymitis. Left testicle 27 × 26 × 32 mm in size, with heterogeneous echogenicity. The color Doppler flow is increased. The epididymis is enlarged and vascularized. Diffuse edema of the subcutaneous cellular tissue in the left hemiscrotal. The right testicle is smaller (18 × 26 × 26 mm) and shows a normal vascularization. (E) Computed tomography of the chest with contrast: axial plane (left) and coronal plane (right). Top (08/04/2021): multiple bilateral pseudonodular ground-glass lung opacities associated with bilateral pleural effusion. Bottom (20 days later): bilateral parenchymal involvement with extensive ground glass areas. In the lower left lobe, there was an increase in the radiodensity of the pre-existing opacities with multiple peripheral consolidations that were not seen in the previous study. Moderate bilateral pleural effusion, which enters through the fissures, had worsened slightly compared to the previous study. Mild pericardial thickening of up to 3 mm.

Figure 2

Anakinra treatment reduces NLRP3 inflammasome activation (A and B) Release of IL-1β from peripheral blood mononuclear cells (A) and percentage of ASC-specking monocytes (B), at baseline or after canonical activation of NLRP3 (LPS+ATP) and pyrin (LPS+TcdB) inflammasome, in the absence/presence of MCC950 from healthy donors (n = 2–3, each dot represents an individual donor, white bars) and the index patient with the KRAS^G12D mutation before anakinra administration (gray bars). (C) Percentage of monocytes with an ASC oligomer from the index patient over the time points examined before and after anakinra treatment. (D) Percentage of monocytes with an ASC oligomer from the index patient after canonical NLRP3 inflammasome activation with LPS+ATP (green) or pyrin inflammasome activation with LPS+TcdB (dark blue) over the time points examined before and after anakinra treatment. (E and F) Release of IL-1β (E) and IL-18 (F) from peripheral blood mononuclear cells, at baseline or treated, as indicated for NLRP3 (LPS+ATP) or pyrin (LPS+TcdB) inflammasome canonical activation, in healthy donors (n = 3, each dot represents an individual donor, white bars) and the index patient during anakinra treatment (green bars). (G) Plasma levels of ASC in the index patient along the time points examined before and after anakinra treatment. (H) Concentration of IL-1 family cytokines in the plasma of the CMML KRAS^G12D index patient in response to anakinra. For (A)–(F), are represented as mean ± SEM, and each dot represents an individual donor.

Figure 3

Monocytes from CMML KRAS^mut patients present a constitutive inflammasome activation (A) Percentage of ASC-specking monocytes in healthy controls (white bars), CMML patients without KRAS mutation (KRAS^wt, blue bars), and CMML patients with a KRAS mutation (KRAS^mut, orange bars) at baseline or treated as indicated for NLRP3 (LPS+ATP) or pyrin (LPS+TcdB) inflammasome activation. (B and C) Release of IL-1β from peripheral blood mononuclear cells (PBMCs) (B) and the formation of ASC specks in monocytes (C) in CMML KRAS^mut patients at baseline or after the indicated stimulation or treatment (LPS+ATP and LPS+TcdB for NLRP3 or pyrin inflammasome, respectively), in the absence/presence of MCC950. (B) Fold increase was calculated to control non-stimulated conditions, where the average of the higher value used to calculate fold increase is 1,066.58 pg/mL. (D) Percentage of extracellular LDH from untreated PBMCs from healthy donors (white bar), KRAS^wt patients (blue bar), and KRAS^mut patients (orange bar). Data are normalized to the percentage of monocytes. (E) Plasma concentration of HMGB1 and P2X7 receptor from healthy donors (white bar), KRAS^wt patients (blue bar), and KRAS^mut patients (orange bar). For (A)–(E), data are represented as mean ± SEM; each dot represents an individual patient; ordinary one-way ANOVA test (two-tailed) was used for (A) (∗ compares CMML KRAS^mut vs. healthy controls; † compares CMML KRAS^mut vs. CMML KRAS^wt), and two-tailed t test in (B)–(E). Note that ∗ or †p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗ or ††††p < 0.0001; ns, no significant difference (p > 0.05).

Figure 4

Plasma cytokines in CMML patients (A) Plasma concentration of IL-1α, IL-1β, IL-1RA, IL-18, IL-12 p40 and IL-12 p70, TNF-α, IL-6, and IL-8. Data are represented as mean ± SEM. Each dot represents an individual patient. Mann-Whitney U test (two-tailed) was used to compare between groups: KRAS^mut (n = 9) and KRAS^wt patients (n = 8), healthy controls (n = 9), and patients with sepsis (n = 5, as control of acute inflammation). Outliers were detected in the datasets by the Grubbs’ test (α = 0.001). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (B) Plasma cytokine concentration showing significant differences observed when comparing CMML KRAS^wt vs. healthy controls, CMML KRAS^mut vs. healthy controls, and CMML KRAS^mut vs. CMML KRAS^wt (Volcano plots). The x axis represents log2 (fold change) and the y axis represents the negative log (p value). Statistically increased (red) was considered when p <0.05 and fold change >1, and statistically decreased (blue) was considered when p <0.05 and fold change <1. Each dot represents a cytokine, and the relevant ones are indicated. IL-2, IL-10, and GM-CSF were not detected in most patients.