The role of fusion genes and cancer driver genes in malignant transformation has traditionally been explored using transgenic or chimeric mouse models. It has been challenging to develop models that fully resemble the characteristics and morphology of human cancers. This applies to anaplastic large-cell lymphoma (ALCL), a malignancy classified as a peripheral T-cell lymphoma. It is still unclear at which stage of T-cell development ALCL can occur, as well as the early molecular events required for malignant transformation. In this issue of Cancer Research, Pawlicki and colleagues introduced the NPM–ALK fusion gene and mutant variants into primary T cells from healthy donors. By monitoring transduced T-cell clones over time, they demonstrated that transformed T cells undergo a progressive loss of T-cell identity accompanied with upregulation of epithelial-to-mesenchymal transition program and reemergence of an immature, thymic profile. Introduction of NPM–ALK was, however, not sufficient to convert healthy T cells to malignant clones, as this process required activation of T-cell receptor signaling. The study sets the stage for modeling early genetic changes in human tumors.

See related article by Pawlicki et al., p. 3241

Anaplastic large-cell lymphoma (ALCL) is a rare malignancy of peripheral T-cell lymphoma, with highest prevalence in children and young adults. A mature CD4+ T cell has traditionally been considered as cell of origin, although ALCL cells have little resemblance to normal T cells. Morphologically, ALCL cells consist of large atypical cells expressing the TNF receptor family member CD30, but with reduced or no expression of the key T-cell identity markers, CD3 and T-cell receptor (TCR; ref. 1). According to the 2016 World Health Organization classification, ALCL is classified into two clinically distinct entities, anaplastic lymphoma kinase positive (ALK+) and ALK negative. Aberrant activation of the receptor tyrosine kinase ALK is a strong oncogene in several different types of cancer and can occur either due to activating mutations or chromosomal translocations. Whereas a variety of fusion partners have been identified and characterized, they all have in common transcriptional regulation driven by regulatory regions of the partner gene and a constitutive activation of the ALK kinase domain upon dimerization of fusion proteins (2). The majority of ALK+ ALCL cases carry the t(2;5)(p23;q35) translocation, causing a gene fusion between the nucleolar protein nucleophosmin (NPM) and the catalytic domains of ALK. NPM–ALK is a key oncogenic driver in ALK+ ALCL by activating multiple signaling pathways, including STAT3, STAT5b, MAPK, and PI3K/mTOR, thereby hijacking physiologic pathways that promote survival and proliferation of T cells (1). Introduction of NPM–ALK is, however, likely not sufficient to transform healthy T cells to become malignant, as the t(2;5) translocation can be detected in cord blood of healthy newborns (3).

It has been challenging to develop murine models that recapitulate the clinical and phenotypical characteristics of human ALCL. Transgenic mice with expression of NPM–ALK restricted to CD4+ T cells develop T-cell lymphoma and plasma cell neoplasms with high penetrance (4). However, these murine tumors are restricted to the thymus, whereas ALCL cells in humans mostly localize to the periphery. Normal thymocytes undergo positive and negative selection in the thymus, and successful rearrangement of the TCR is required for positive selection and transition from the CD4+CD8+ double positive stage and later egress from the thymus to the circulation as a naïve T cell. The majority of patient-derived ALCL cells have in-frame rearrangement of the TCR α chain, suggesting requirement for a functional TCR at some point during ALK-driven tumorigenesis. A recent study provided new insight in the putative role of a functional TCR for NPM–ALK-driven tumor formation (5). By crossing CD4/NPM–ALK mice with TCR transgenic mice, the offsprings developed peripheral T-cell lymphoma, usually with loss of surface TCR expression, hence mimicking human ALCL. However, in the presence of specific antigen, these mice did not develop peripheral T-cell tumors. These results suggest that expression of TCR is needed for thymic egression, but also that continuous TCR signaling is not compatible with ALK-driven malignancy. Whether human ALCL arises from an early precursor cell in the thymus or from reprogramming of mature CD4+ T cells in the periphery remains an unresolved question.

In this issue of Cancer Research, Pawlicki and colleagues decipher the molecular dynamics of human T-cell transformation (6). In previous work, this group showed that lentivirus-induced NPM–ALK expression in primary mature CD4+ T cells caused immortalization and induced morphologic and phenotypical changes, indistinguishable from patient-derived NPM–ALK+ ALCL cells (7). Here, using their model of NPM–ALK-driven CD4+ T-cell transformation to explore the mechanisms of oncogenesis, the authors demonstrate that early T-cell transformation is accompanied by dynamic and profound transcriptional changes (6). Gene set enrichment analysis of gene expression profiles distinguishing activated NPM–ALK+ CD4+ T cells from T cells transduced with a kinase-defective NPM–ALK indicated a progressive attenuation of T-cell effector programs and the upregulation of epithelial-to-mesenchymal transition (EMT) and pluripotency signatures. The authors noted a stepwise progression, starting with a repression of cMYC-targeted genes, followed by the upregulation of STAT3 signaling, and a gradual loss of T-cell identity and effector functions as the final step of the transformation process. These findings indicate that NPM–ALK induces a dedifferentiation of mature CD4+ T cells toward a more progenitor-like state. The latter observation was strongly supported by the introduction of a triple point tyrosine NPM–ALK mutant (NPM–ALKTrpM), unique among a plethora of tyrosine mutants generated by the authors, which did not alter T-cell–specific transcription factors nor induced EMT initiation and had lost the capacity to transform primary CD4+ T cells.

Accordingly, new evidence has emerged from this study to support the hypothesis that the progenitor phenotype of NPM–ALK+ ALCL cells is orchestrated by NPM–ALK fusion rather than a putative thymic origin of ALCL neoplasms. Furthermore, the authors took advantage of the NPM–ALKTrpM model to investigate signaling complexes critical for T-cell transformation. In addition to adaptor molecules from the Shc and Grb families, the authors identified new interacting partners of NPM–ALK, including AGHGEF5 and UBASH3B, involved in promoting EMT. Finally, even though NPM–ALK fusion is the oncogenic hallmark of ALCL, it is not sufficient to drive T-cell transformation. Pawlicki and colleagues moved their findings forward and uncovered the requirement for TCR engagement to achieve T-cell transformation (6). An early cooperation between TCR-generated signals upon antigen recognition and signaling cascades activated by NPM–ALK fusion is essential for the homeostatic control to fail and for otherwise resistant T cells to transform. The latter finding addresses the question of why not all T cells bearing NPM–ALK fusion acquire a malignant phenotype.

In line with the findings by Pawlicki and colleagues, recent studies have gathered further evidence of the thymic progenitor–like reprogramming of NPM–ALK+ ALCL. In analysis of DNA methylomes, primary ALCL tumor cells and NPM–ALK-transformed CD4+ T cells clustered together and distant from healthy T cells. More importantly, their methylomes shared close similarity with that of thymic progenitors, representing the earliest stages of T-cell development (8). Transcriptional patterns further linked NPM–ALK-transformed CD4+ T cells and patient-derived ALCL cells to early thymic precursors, supported by the expression of pluripotency-associated transcription factors, OCT4, SOX2, and NANOG. While NPM–ALK is central in the process of T-cell reprogramming to achieve its oncogenic potential, it needs to coordinate an extensive network of intracellular signaling cascades. Recent mechanistic studies highlight STAT3 as a key downstream mediator of NPM–ALK oncogenic activity. Constitutively, acetylated STAT3 in ALK+ ALCL associates with the transcriptional activator Sin3 and jointly lead to epigenetic silencing of tumor suppressor genes (9). Candidate tumor suppressors silenced because of ALK oncogenic activity include TCR signaling regulators, WASP and WIP. Their expression is selectively downregulated in patient-derived ALCL, but retained in other T-cell lymphomas dependent on tonic TCR signaling (10). The downregulation of WASP and WIP provides a biological advantage to tumor cells by increasing MAPK signaling and thus contributes to an accelerated pathogenesis of ALCL.

It is remarkable how NPM–ALK could drive the profound remodeling of the epigenetic, transcriptional, phenotypical, and morphologic profile of mature T cells. This new understanding of reprogramming was facilitated by the development of a robust model of lentivirus-induced NPM–ALK transformation of healthy T cells. The cell of origin of ALCL has been an open question due to a lack of model systems that reflected human disease and limited patient material from which to divulge information. Transgenic mouse models have been useful to discover important aspects of malignant transformation, however, they recapitulate only some features of the ALCL malignancy. On the other hand, patient material comes from already advanced tumors, which precludes study of the early stages of tumorigenesis. Therefore, recreating the process of cell transformation is challenging and having a robust model is a prerequisite to gain a deeper knowledge of the genetic and epigenetic rearrangements that drive tumor transformation. As Pawlicki and colleagues demonstrated, the development of de novo models of tumor cell transformation announces a new era of improved understanding of the complex processes of human tumorigenesis.

No disclosures were reported.

The work in the authors' laboratory was supported by the Foundation KG Jebsen (#19, Centre for B-cell Malignancies) and grant from The Norwegian Cancer Society (182694- precision medicine in B-cell lymphoma).

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