Oncogenic Potential and Sensitivity to Menin Inhibitors of a Novel Fusion KAT6B::NUTM2B in Congenital Myeloid Sarcoma with Bone Marrow Involvement
Authors: Voss Rebecca , Qi Wenqing , Liu Yen-Chun , Ma Jing , Browne La'Ron , Khanlari Mahsa , Barajas Juan M , Thomas Melvin , Pastor Loyola Victor B , Cao Hui , Karol Seth E. , Rubnitz Jeffrey E. , Klco Jeffery M , Wang Lu 
Published: December 2024

Introduction

Congenital leukemia is a rare disease that may present as myeloid sarcoma, with or without bone marrow involvement. Though some cases show spontaneous remission, outcome is generally poor. Acute myeloid leukemia (AML) is the most common subtype, more than half of which are associated with rearrangements involving KMT2A or KAT6A. Here, we report a novel KAT6B::NUTM2B (KN) fusion involving the KAT6A paralog, KAT6B, identified in an infant with AML who presented with congenital myeloid sarcoma and bone marrow involvement. KAT6A/B form a complex facilitating histone acetylation, they cooperatively promote transcription of genes regulating hematopoiesis. Rearrangements involving these genes lead to the activation of oncogenes such as the HOXA cluster, promoting leukemogenesis. Our in vitro study demonstrates oncogenic potential of KN fusions and shows promising results for a therapeutic intervention using a menin inhibitor.

Methods

Bone marrow and skin biopsy samples were evaluated by routine clinical morphologic and immunophenotyping profiling. The novel KAT6B::NUTM2B fusion was detected by whole genome (WGS) and whole transcriptome sequencing (WTS) in both skin lesion and bone marrow specimens of a 9-day-old infant with congenital myeloid sarcoma. The transforming properties of KAT6B::NUTM2B were studied in vitro by retroviral transduction of C57BL/6J murine bone marrow hematopoietic stem and progenitor cells (mHSPC) and conducting a serial replating Colony Forming Unit (CFU) assay. The KN fusion construct and controls (empty vector, KAT6B and NUTM2B wild-type) were cloned into MSCV-IRES-GFP or MSCV-IRES-mCherry vectors and used to transfect HEK293T cells to generate retrovirus. Mouse HSPCs were transduced, sorted for GFP/mCherry positive cells and plated as triplicates in methylcellulose-based semisolid medium (M3434). Colonies were counted every 7 days, harvested and serially replated. To test the sensitivity to the menin inhibitor SNDX-5613, varying concentrations of SNDX-5613 were added to CFU medium.

Results

Clinical flow cytometry of the bone marrow sample revealed an abnormal cell population (20.17%) positive for CD14 (subset), CD11c, CD64, CD33, CD11b, HLA-DR, CD123, and MPO and negative for CD34, CD117, CD133, NG2, CD19, and CD3 (surface and cytoplasmic). Increased morphologic blasts along with the immunophenotypic findings established a diagnosis of acute myeloid leukemia with monocytic differentiation. A biopsy of the patient's concurrent skin lesion confirmed the diagnosis of myeloid sarcoma.

WGS analysis of the patient's tumor samples demonstrated a cryptic interstitial deletion (~4.8Mb) at 10q22 resulting in a fusion between KAT6B and NUTM2B. Located on 10q22.2 and 10q22.3, respectively, both genes run in the same direction. WTS confirmed an in-frame fusion transcript between KAT6B exon 17 and NUTM2B exon 2, preserving the histone acetyltransferase domain of KAT6B and nuclear localization sequence of NUTM2B. Transcriptome profiling with comparison to known AML subtypes by uniform manifold approximation and projection (UMAP) showed the case clustering with KAT6A or KMT2A-fusion positive AMLs. Analysis of HOX gene expression revealed overexpression of HOXA9 and HOXA10.

When introduced into mHSPCs, KN-positive cells showed a substantial and constant increase in colony formation and serial replating beyond 4 weeks compared to the controls. Colony formation for controls drastically decreased after week 2. Considering the observed HOXA9/10 activation, KN-positive cells were treated with SNDX-5613 or DMSO as control. By week one, colony formation decreased by 30%, 20% and 50% for 0.25 µM, 1 µM, and 1.5 µM SNDX, respectively. By week 2, after continued treatment with SNDX/DMSO, the effect was more pronounced with a 70%, 80% and 90% decrease for the same concentrations.

Conclusion

In summary, we are the first to report a novel oncogenic fusion, KAT6B::NUTM2B, in a case of congenital myeloid sarcoma with bone marrow involvement. Whole transcriptome analysis of the KN-positive case demonstrated a transcriptomic profile similar to that of KAT6A and KMT2A fusions and activation of HOXA9/10 genes. Furthermore, our in vitro study indicated a dramatic proliferative potential and oncogenic transformation of KN-positive cells and sensitivity to menin inhibitor, suggesting a promising new therapeutic approach for KAT6B-associated fusions.