LNK/ SH2B3 as a novel driver in juvenile myelomonocytic leukemia

Abstract

Mutations in five canonical Ras pathway genes (NF1, NRAS, KRAS, PTPN11 and CBL) are detected in nearly 90% of patients with juvenile myelomonocytic leukemia (JMML), a frequently fatal malignant neoplasm of early childhood. In this report, we describe seven patients diagnosed with SH2B3-mutated JMML, including five patients who were found to have initiating, loss-of-function mutations in the gene. SH2B3 encodes the adaptor protein LNK, a negative regulator of normal hematopoiesis upstream of the Ras pathway. These mutations were identified to be germline, somatic or a combination of both. Loss of function of LNK, which has been observed in other myeloid malignancies, results in abnormal proliferation of hematopoietic cells due to cytokine hypersensitivity and activation of the JAK/STAT signaling pathway. In vitro studies of induced pluripotent stem cell-derived JMML-like hematopoietic progenitor cells also demonstrated sensitivity of SH2B3-mutated hematopoietic progenitor cells to JAK inhibition. Lastly, we describe two patients with JMML and SH2B3 mutations who were treated with the JAK1/2 inhibitor ruxolitinib. This report expands the spectrum of initiating mutations in JMML and raises the possibility of targeting the JAK/STAT pathway in patients with SH2B3 mutations.

Figure 1.

Schematic overview of SH2B3 including the location of both primary and secondary mutations described in juvenile myelomonocytic leukemia. The top row shows the mutations of the seven novel patients reported here; the bottom row shows the location of the mutations previously reported by our group. Mutations that are considered to initiate juvenile myelomonocytic leukemia are highlighted in red boxes. Alterations that co-exist with a PTPN11 mutation are displayed with a dashed line. DD: dimerization domain; PH: pleckstrin homology domain; SH2; Src homology 2 domain; n/a: not available.

Figure 2.

SH2B3-mutated hematopoietic progenitor cells are more sensitive to JAK inhibitor therapy. (A) Linear regression plot of a high throughput drug discovery screen comparing drug inhibition of PTPN11/SH2B3 double-mutant hematopoietic progenitor cells (HPC) versus PTPN11 single-mutant HPC. The top ten hits that inhibited growth of double-mutant HPC to a greater extent than that of single-mutant HPC include two JAK inhibitors: momelotinib and CEP-33779. (B) Cell viability assay readout 72 hours after exposing two different induced pluripotent stem cell-derived HPC lines to ruxolitinib or momelotinib. Data for tofacitinib are not shown. HPC: hematopoietic progenitor cells.

Figure 3.

Phylogenetic tree in a patient with juvenile myelomonocytic leukemia. The phylogenetic tree at diagnosis in patient UPN2861 was inferred from single-cell sequencing and single-cell inference of tumor evolution (SCITE), a probabilistic model using a flexible Markov-chain Monte Carlo algorithm.37 A heterozygous SH2B3 p.M268I was the initiating mutation, which then branched into a PTPN11 population and a homozygous SH2B3 population. The PTPN11 population finally branched into WT1 and IKZF1 clones. HET: heterozygous; HOM: homozygous.

Figure 4.

Molecular response of patient UPN3037 who harbored a PTPN11 and two SH2B3 mutations at diagnosis. Following 10 days of ruxolitinib monotherapy, the SH2B3 mutation at codon 523 was no longer detectable, and the allele frequency of the SH2B3 mutation at codon 408 decreased from 38% to 11%. VAF: variant allele frequency.