Mycosis fungoides (MF) is a slowly progressive cutaneous T-cell lymphoma (CTCL) for which there is no cure. In the early plaque stage, the disease is indolent, but development of tumors heralds an increased risk of metastasis and death. Previous research into the genomic landscape of CTCL revealed a complex pattern of >50 driver mutations implicated in more than a dozen signaling pathways. However, the genomic mechanisms governing disease progression and treatment resistance remain unknown. Building on our previous discovery of the clonotypic heterogeneity of MF, we hypothesized that this lymphoma does not progress in a linear fashion as currently thought but comprises heterogeneous mutational subclones. We sequenced exomes of 49 cases of MF and identified 28 previously unreported putative driver genes. MF exhibited extensive intratumoral heterogeneity (ITH) of a median of 6 subclones showing a branched phylogenetic relationship pattern. Stage progression was correlated with an increase in ITH and redistribution of mutations from stem to clades. The pattern of clonal driver mutations was highly variable, with no consistent mutations among patients. Similar intratumoral heterogeneity was detected in leukemic CTCL (Sézary syndrome). Based on these findings, we propose a model of MF pathogenesis comprising divergent evolution of cancer subclones and discuss how ITH affects the efficacy of targeted drug therapies and immunotherapies for CTCL.

Iyer and colleagues used deep sequencing of T-cell receptor genes to demonstrate clonal heterogeneity of mycosis fungoides, with repeated seeding of disparate clones from the blood.

Mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, is believed to represent a clonal expansion of a transformed skin-resident memory T cell. T-cell receptor (TCR) clonality (ie, identical sequences of rearranged TCRα, TCRβ, and TCRγ), the key premise of this hypothesis, has been difficult to document conclusively because malignant cells are not readily distinguishable from the tumor-infiltrating reactive lymphocytes that contribute to the TCR clonotypic repertoire of MF. Here, we have successfully adopted targeted whole-exome sequencing (WES) to identify the repertoire of rearranged TCR genes in tumor-enriched samples from patients with MF. Although some of the investigated MF biopsies had the expected frequency of monoclonal rearrangements of TCRγ corresponding to that of tumor cells, the majority of the samples presented multiple TCRγ, TCRα, and TCRβ clonotypes by WES. Our findings are compatible with the model in which the initial malignant transformation in MF does not occur in mature memory T cells but rather at the level of T-lymphocyte progenitors before TCRβ or TCRα rearrangements. We have also shown that WES can be combined with whole-transcriptome sequencing in the same sample, which enables comprehensive characterization of the TCR repertoire in relation to tumor content. WES/whole-transcriptome sequencing might be applicable to other types of T-cell lymphomas to determine clonal dominance and clonotypic heterogeneity in these malignancies.

Mature T-cell lymphomas (TCLs) are rare, clinically heterogeneous hematologic cancers with high medical need. TCLs have an inferior prognosis which is attributed to poor understanding of their pathogenesis. On the basis of phenotypic similarities between normal and neoplastic lymphocytes, it has been assumed that TCLs develop in the periphery, directly from various subtypes of normal T cells. To address the debated question of the cell of origin in TCLs, we attempted to identify the highly variable complementarity-determining regions (CDRs) of T-cell receptors (TCRs) to trace the clonal history of the T cells. We have collected previously published whole-genome, whole-exome, and whole-transcriptome sequencing data from 574 patients with TCL. TCR clonotypes were identified by de novo assembly of CDR3 regions of TCRα, TCRβ, and TCRγ. We have found that the vast majority of TCLs are clonotypically oligoclonal, although the pattern of oligoclonality varied. Anaplastic large-cell lymphoma was the most diverse comprising multiple clonotypes of TCRα, TCRβ, and TCRγ, whereas adult TCL or leukemia and peripheral TCLs often showed monoclonality for TCRβ and TCRγ but had diverse TCRα clonotypes. These patterns of rearrangements indicated that TCLs are initiated at the level of the lymphoid precursor. In keeping with this hypothesis, TCR rearrangements in TCLs resembled the pattern seen in the human thymus, which showed biased usage of V (variable) and J (joining) segments of high combinatorial probability resulting in recurrent public CDR3 sequences shared across unrelated patients and different clinical TCL entities. Clonotypically diverse initiating cells may seed target tissues that are then responsible for disease relapses after therapy.

Ultraviolet (UV) radiation is a strong environmental carcinogen responsible for the pathogenesis of most skin cancers, including malignant melanoma (MM) and non-melanoma (keratinocyte) skin cancers. The carcinogenic role of UV was firmly established based on epidemiological evidence and molecular findings of the characteristic mutation signatures which occur during the excision repair of cyclobutane pyrimidine dimers and 6,4-photoproducts. The role of UV in the pathogenesis of mycosis fungoides (MF), the most common type of primary cutaneous T-cell lymphoma, remains controversial. Here, we performed whole-exome sequencing of 61 samples of MF cells microdissected from cutaneous lesions, and compared their mutational signatures to 340 MMs. The vast majority of MM mutations had a typical UV mutational signature (SBS 7, SBS 38, or DSB 1), underscoring the key role of ultraviolet as a mutagen. In contrast, the SBS 7 signature in MF comprised < 5% of all mutations. SBS 7 was higher in the intraepidermal MF cells (when compared to the dermal cells) and in the cells from tumors as compared to that in early-stage plaques. In conclusion, our data do not support the pathogenic role of UV in the pathogenesis of MF and suggest that the UV mutations are the result of the cumulative environmental ultraviolet exposure of cutaneous lesions rather than an early mutagenic event.

Acute myeloid leukemia (AML) is a hematologic malignancy with limited treatment options and a high likelihood of recurrence after chemotherapy. We studied N-myristoylation, the myristate modification of proteins linked to survival signaling and metabolism, as a potential therapeutic target for AML. N-myristoylation is catalyzed by two N-myristoyltransferases (NMT), NMT1 and NMT2, with varying expressions in AML cell lines and patient samples. We identified NMT2 expression as a marker for survival of patients with AML, and low NMT2 expression was associated with poor outcomes. We used the first-in-class pan-NMT inhibitor, zelenirstat, to investigate the role of N-myristoylation in AML. Zelenirstat effectively inhibits myristoylation in AML cell lines and patient samples, leading to degradation of Src family kinases, induction of endoplasmic reticulum stress, apoptosis, and cell death. Zelenirstat was well tolerated in vivo and reduced the leukemic burden in an ectopic AML cell line and in multiple orthotopic AML patient-derived xenograft models. The leukemia stem cell–enriched fractions of the hierarchical OCI-AML22 model were highly sensitive to myristoylation inhibition. Zelenirstat also impairs mitochondrial complex I and oxidative phosphorylation, which are critical for leukemia stem cell survival. These findings suggest that targeting N-myristoylation with zelenirstat represents a novel therapeutic approach for AML, with promise in patients with currently poor outcomes.