KLF5 Is a Key Regulator of IMiD-Induced Neutropenia

Although immunomodulatory drugs (IMiDs, lenalidomide [LEN] & pomalidomide [POM]) have had a huge impact on the therapeutic landscape in multiple myeloma (MM), IMiD-induced neutropenia remains a clinical challenge. Understanding the molecular basis of IMiD-associated myelosuppression could improve the rational design of novel agents which mitigate this important side effect whilst retaining therapeutic efficacy against MM cells. We have previously established an experimental system to carry out single cell multiomic analyses of neutrophil differentiation ex vivo, allowing us to study the impact of exposure to IMiDs, which caused a maturation impairment and decrease in the abundance of differentiating granulocytes¹. These findings aligned with previous clinical observations of a myeloid maturation arrest which is associated with IMiD-induced neutropenia in MM patients, with Cereblon (CRBN)-driven IKZF1 degradation previously implicated as the underlying cause. However, restoration of IKZF1 expression only partially rescues the IMiD-associated neutrophil maturation arrest, suggesting that other mechanisms might also contribute.

The purpose of the current study was to interrogate our single cell multiomic dataset to characterise the impact of IMiDs on the dynamic genome regulatory landscape during neutrophil development and thereby identify putative novel neo-substrates that might contribute to myelosuppression.

Analysis of single cell RNA seq (n=3 donors and 111,109 cells) and multiome datasets (n=3 donors and 47,452 cells) of ex vivo neutrophil differentiation and its perturbation by IMiDs, allowed us to construct a gene regulatory map to identify putative driver genes implicated in the pathobiology of IMiD-mediated neutrophil maturation arrest. Combined gene expression and chromatin accessibility analysis based on the detection of 330,334 peak-to-gene links correlated chromatin architecture to neutrophil terminal differentiation potential. Myeloid progenitors contained relaxed chromatin, whilst IMiD-associated abnormal myeloid precursors states were characterized by premature chromatin compaction (as reflected by a 30% decline in filtered peaks) and priming towards a pro-apoptotic state, despite bearing gene expression profiles largely reminiscent of intact neutrophils and precursors. This finding was corroborated by the reduction of accessible enhancer loci (4.5-14.8%), suggesting a repression of transcriptional activity in the treated populations.

To enrich for candidate transcription factors, we selected motifs that were not altered in expression at the transcript level in the granulocyte-monocyte progenitor and metamyelocyte clusters but showed marked IMiD-induced changes in their associated regulons at the transcript level. Furthermore, ATAC-seq footprinting allowed us to infer altered transcription factor binding dynamics in the same cells. As expected, IKZF1 met these criteria alongside SPI1,CEBPA, ZBTB7A, ZBTB7B, KLF5, PPARA, PPARG additional candidate genes.

To further prioritise candidate neo-substrates, we intersected our data with an extensive proteomic screening to enrich for CRBN-interacting small molecules. Amongst the genes overlapping in both our multiomic analysis and proteomic screen was KLF5, a known transcription factor involved in the regulation of neutrophil differentiation in mice. Moreover, we also identified downregulation of KLF5 binding partners C/EBPβ and C/EBPδ and the downstream target PPARG alongside a gradual switch from the overarching (during normal granulopoiesis) glycolytic metabolic machinery towards increased fatty acid oxidation. This surrogate pathway has been previously associated with skewed neutrophil effector functions and described in genome-wide association studies (GWAS) focusing on abnormal neutrophil counts. Intracellular FACS analysis confirmed rapid KLF5 degradation upon exposure to IMiDs, confirming that this is a likely IMiD-associated CRBN neo-substrate. Proximity assays, rescue studies and conformation capture assays to dissect the relevant key regulatory elements are underway.

Our study has identified KLF5 as a putative novel regulator of IMiD-induced neutropenia which might inform the rational design of new therapeutic agents to mitigate this problematic side effect of widely used drugs in MM.

1.https://doi.org/10.1182/blood-2023-182041