Degradation of Ikaros Induces Neutropenia through Altered Transcriptional Programming across Multiple Stages of Neutrophil Development and Maturation

Introduction: Neutrophils play a vital role in the innate immune system, for surveillance and response to infections. Derived from hematopoietic stem cells in bone marrow, myeloid progenitor cells undergo maturation stages before exiting as mature neutrophils. Certain drugs in hematologic malignancy treatment, including chemotherapy, can lead to neutropenia by affecting myeloid progenitor viability. Immunomodulatory drugs (IMiDs) like lenalidomide can also cause neutropenia without the toxic effects seen with cytotoxic agents. Clinically, neutropenia can be managed through growth factor support or alternative drug administration schedules. The molecular mechanism of IMiDs involves targeting CRBN, a substrate receptor in the CRL4^CRBN E3 ligase complex, leading to the degradation of neosubstrates like Ikaros (IKZF1). Ikaros, a transcription factor crucial for hematopoiesis, plays a significant role in myeloid development. Using a humanized CRBN mouse model (hCRBN) and defined flow sorted progenitor populations, we evaluate how Ikaros degradation impairs granulopoiesis and investigate the transcriptional networks regulated by Ikaros during neutrophil maturation.

Methods: Homozygous hCRBN mice were treated daily with Ikaros degrader (CC-548) via oral gavage until days 7, 14 and 21, when mice were euthanized. Bone marrow cells were collected for parallel flow cytometric and multiple population gated RNAseq analyses. We performed low input RNAseq using SMARTseq platform on myelopoiesis specific stage defining populations such as multi-potent progenitors, lineage-committed progenitors, pre-myelocytes, immature neutrophils and mature neutrophils.

Results: After an initial elevated population across multiple cell progenitor populations at day 7, deep immunophenotypic analyses showed an acute decrease in early stage LSK progenitors including MMP2 and MMP3, in tandem with declines in pre-neutrophil and immature neutrophil populations. Over time, this resulted in systemic neutropenia and higher numbers of immature neutrophils in circulation, which is linked to IKZF1 degradation. RNAseq analysis on presorted sub-populations revealed differential expression of a relatively small, conserved set of genes (n=184) spread across multiple pathways, especially in cell cycle, metabolic and autophagy modulation. Using inference algorithms with regulatory transcription factor (TF) networks defined by differentially expressed genes, we identified TF networks such as PU.1, Myc and E2F family TFs as common regulatory networks affected by treatment with CC-548 compared to vehicle control in multi-potent progenitors, lineage committed progenitors (e.g. LK and LSK cells) and pre-myelocytes. The Myc and PU.1 TF networks remain common across time points from day 7 to 21 and across all measured populations, but some TFs linked to stem cell renewal were downmodulated across early precursor populations depending on duration of dosing (e.g. Gata2, Hlf, Rara, Sox6). However, myeloid lineage linked TFs and genes showed differential effects across LSK and LK populations following CC-548 treatment. These include Irf8, Sox4, Cebpe, Cst7 and Csf1r. Similarly, key cell cycle and stress related genes in LSK cells, such as CDKN1a (p21), KLF4, Gstm2, Mgst2, Ptger2, Ctsg were all upregulated in a kinetic manner as a consequence of homeostatic responses with CC-548 dosing. Additionally, genes involved in medullary retention of these precursor cells, such as SelP (P-selectin) were upregulated initially, but decreased with extended dosing. Other linked genes were more subtly modulated, including cyclins CCND1 and CCNE1. Both immature & mature neutrophils showed deregulation of TF networks affecting cell cycle, granule production, RNA/splicing, and chemotaxis. Further computational analyses to build more comprehensive gene regulatory networks are underway to connect impairment of neutrophil development to degradation of Ikaros via CRBN mediated molecules.

Conclusion: These studies delineate regulatory networks affected by IKZF1 degradation across various stages of granulopoiesis, which ultimately leads to neutropenia. The findings underscore the importance of Ikaros as a master regulator of transcriptional regulation in myeloid differentiation. Understanding this biology may lead to strategies for neutropenia mitigation via new agents or dose/schedule considerations.