Overactivation of the NLRP3 Inflammasome in Chronic Myelomonocytic Leukemia KRAS Mutated Patients Can be Detected By the Apoptosis-Associated Speck-like Protein (ASC) and Reverted By IL1β Inhibitors

It has been recently shown that RAS mutations, which occur in 11-38% of Chronic Myelomonocytic Leukemia (CMML), do not only act via RAS/MEK/ERK signaling, but contribute to the disease through NLRP3 inflammasome activation (Hamarsheh, Nat Comm 2020). Despite a therapeutic approach based on NLRP3/IL1β axis blockade, as bring to a stem cell transplantation (SCT) has been proposed, data on the efficacy of IL1β inhibitors in hematopoietic neoplasms is limited.

A 55 year old man with previous autoinflammatory episodes (constrictive pericarditis) was diagnosed on September 2020 of CMML-1 KRAS ^G12D (Inter-2). Due to worsening (orchiepidedymitis, pneumonitis, cellulitis), and the impossibility of performing an SCT at that time, on December 02 2020 he started anakinra (a IL1β receptor antagonist) with good response. Due to new episodes of autoinflammation, anakinra was discontinued (12 April 2021) with severe clinical worsening (heart failure) and no response to diuretic/corticosteroid. After anakinra was restarted (04 May 2021), a progressive improvement was seen, allowing a successful pericardiectomy before an SCT. We obtained blood samples from this patient (at different times) and plasma and whole blood samples from 11 and 5 other CMML KRAS ^mut patients, respectively. We also included CMML patients without KRAS mutations (KRAS ^wt) (n=8), with sepsis (n=5) and healthy individuals (n=9).

Plasma levels of 15 inflammatory cytokines associated with NLRP3 inflammasome and NFkB pathways were measured using a customized MILLIPLEX ^® kit. The inflammasome marker activation assays were conducted as previously published (Martínez García JJ, Nature Comm 2019).

Compared to healthy controls, KRAS ^wt CMML patients did not show differences in any cytokine tested, except IL6, while KRAS ^mut patients showed significantly higher levels of IL1α, IL1ra, IL18, IL12p40 (associated with NLRP3 inflammasome), IL6, IL8 (associated with NFkB pathway) and M-CSF (Fig. 1A B). Compared to KRAS ^wt CMML patients, those with KRAS ^mut showed higher levels of cytokines associated with both the NLRP3 and NFkB pathways, reaching statistical significance for those related with NLRP3 inflammasome. We also observed changes in inflammasome related cytokines before and after anakinra (Table 1).

This cytokine profile in the plasma made us analyze the oligomerization of ASC as a marker of inflammasome activation in monocytes of KRAS ^mut CMML. We found that in all cases of KRAS ^mut CMML patients around 30 to 80% of monocytes presented oligomers of ASC measured by the time of flight assay, while in healthy donors and KRAS ^wt CMML patients, ASC oligomerization occurred upon NLRP3 inflammasome activation with lipopolysaccharide (LPS) + ATP or Pyrin inflammasome activation with LPS and Clostridium difficile B toxin (TcdB) (Fig. 2A). Ex vivo activation of PBMCs from KRAS ^mut CMML patients showed that despite the high percentage of cells with ASC oligomers, very low levels of IL1b released from these cells, even when NLRP3 was activated with LPS+ATP (Fig. 2B), suggesting that this inflammasome is activated in vivo and could not be further activated ex vivo. As control, Pyrin inflammasome activation in PBMCs from KRAS ^mut CMML was able to induce IL1b release similarly to healthy controls (Fig. 2B).

We then found that anakinra treatment of the KRAS ^mut CMML patient followed in this study, resulted in a decrease of the percentage of monocytes with basal active inflammasomes (Fig. 2C). A little ex vivo activation of the NLRP3 inflammasome was obtained when cells were treated with LPS+ATP, while Pyrin inflammasome was activated at normal levels after LPS+TcdB treatment (Fig. 2D). The inflammasome basal activation increased in the monocytes of the KRAS ^mut CMML patient after anakinra withdraw and during clinical deterioration and restarting anakinra (second arrow) decreased the basal percentage of monocytes with ASC oligomers (Fig. 2C). Since ASC oligomers are associated to pyroptosis via caspase 1 activation and gasdermin D processing, we then analyzed pyroptotic markers in the plasma of the patient during the time. ASC was increased when monocytes presented elevated percentage of ASC oligomers (Fig. 2E), suggesting that ASC detection could be a promising biomarker.

Overall, we show that, in vivo, the NLRP3 inflammasome activation of KRAS ^mut CMML patients may revert with IL1β blockers. ASC could identify those candidates to receive this therapy.

PI18/00316

Figure 1

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Figure 1

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