Inflammasome and Autophagy Activation In Symptomatic Multiple Myeloma Could Predict Response To Thalidomide-Based Chemotherapy
Authors: V Pericole Fernando , Santos Duarte Adriana Silva , Lazarini Mariana , Olalla Saad Sara Teresinha 
Published: October 2019

Abstract
The inflammasome is a protein complex activated by signals of cellular danger to trigger the innate immune defenses through the maturation of proinflammatory cytokines such as interleukin (IL)1β and IL18. Inflammasomes consist of pattern-recognition sensors (NLR family), adaptors (ASC), and effectors (caspase 1). IL1β initiates feedback loops through the IL1R-MyD88-NFκB pathway. Inflammasome activation promotes adaptive responses and limits reactive oxygen species (ROS) production. Autophagy is a cytoprotective pathway by which the cell sequesters damaged proteins and organelles for lysosomal degradation. The inflammasome is negatively regulated by autophagy and inflammasome activates autophagy. Inflammasome activity may play an important role in several nonmicrobial diseases with chronic inflammation, such as obesity, type 2 diabetes and cancer.
We collected a cohort of diagnostic samples of total bone marrow (BM) from 31 MM patients, 3 plasma cell leukemia (PCL) patients and 9 healthy bone marrow donors (HD), together with clinical data. Four smoldering MM, 27 symptomatic MM and 3 PCL (2 primary and 1 secondary) were included. According to ISS, six patients were ISS I, 9 patients were ISS II and 16 were ISS III. We also stratified 18 of our symptomatic MM into groups according to response (CR/VGPR, PR, SD/PD). The offered treatment was thalidomide-based triplets.
Essential genes from inflammasome (NLRP3, PYCARD, CASP1), pro-inflammatory interleukins (IL1B, IL18) and autophagy (BECN1, SQSTM1, MAP1LC3B) were verified by q-PCR. Gene expression was compared among subgroups and correlated with clinical data.
CASP1 and PYCARD were increased in MM compared with HD, without differences among ISS subgroups. In PCL cases, CASP1 and PYCARD had lower expression when compared with MM, but did not differ from HD, confirming that PCL did not have inflammasome activation as MM did. NLRP3 was not different among all diagnostic subgroups. IL18 had decreased expression in PCL patients, compared with HD and MM. IL1B was not different among subgroups.
PCL and MM had higher BECN1 expression compared with HD and these differences were also highlighted among ISS subgroups. SQSTM1 had increased expression in PCL, compared with HD and MM, but MM did not differ from HD. MAP1LC3B gene expression was similar among groups. A positive correlation was observed between CASP1 and PYCARD, IL1B and IL18, and between the three autophagic genes (BECN1, SQSTM1 and MAP1LC3B) as expected. BECN1 also correlated with IL1B and with CASP1; CASP1 and PYCARD correlated with BECN1 and SQSTM1, reinforcing the relation between autophagy and inflammasome.
When the clinical data were analyzed, monocyte counts correlated with CASP1 and PYCARD; beta-2-microglobulin levels correlated with CASP1 and with NLRP3 and, finally, leukocyte and neutrophil counts correlated with IL1B, BECN1 and SQSTM1 BM expression levels. Analyzing response to thalidomide-based first line chemotherapy, VGPR/CR responders showed higher diagnostic NLRP3 expression (unpaired t test, P=0.03).
The intricate interplay between autophagy and inflammasome has only recently been elucidated. Multiple myeloma is a B-cell neoplasm with great dependence of BM microenvironment. Inflammatory cells, especially monocytes and macrophages, are the main inflammasome activators, reducing ROS in the BM microenvironment. Autophagy is essential for plasmocytes, due to their high levels of intracellular proteins. Basal levels of autophagy in myeloma are high and are upregulated in response to ER stress and proteasome inhibition, protecting myeloma cells against apoptosis.
Our results confirm the essential role of autophagy activation in myeloma. Interestingly, inflammasome activation in our cohort predicted a better response to thalidomide-based regimens and could be a novel response biomarker. Reinforcing our theory, recently thalidomide was described as an inhibitor of caspase 1 activation. Chronic inflammation is an established cancer hallmark and its role in myeloma pathogenesis seems to be important through inflammasome and autophagy dysfunction during disease progression, possibly creating dependency loops between inflammatory monocytes and neoplastic plasmocyte within the BM microenvironment.

Disclosures:
No relevant conflicts of interest to declare.