Summary A systematic characterization of the genetic alterations driving ALCLs has not been performed. By integrating massive sequencing strategies, we provide a comprehensive characterization of driver genetic alterations (somatic point mutations, copy number alterations, and gene fusions) in ALK− ALCLs. We identified activating mutations of JAK1 and/or STAT3 genes in ∼20% of 155 ALK− ALCLs and demonstrated that 38% of systemic ALK− ALCLs displayed double lesions. Recurrent chimeras combining a transcription factor (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were also discovered in WT JAK1/STAT3 ALK− ALCL. All these aberrations lead to the constitutive activation of the JAK/STAT3 pathway, which was proved oncogenic. Consistently, JAK/STAT3 pathway inhibition impaired cell growth in vitro and in vivo.

Following publication of the manuscript, the authors identified an inadvertent error in the summary. The analysis exploring the presence of activating mutations in JAK1 and/or STAT3 was conducted in a total of 155 ALCLs, of which 88 were systemic ALK À ALCLs. The ''155'' in the sentence ''We identified activating mutations of JAK1 and/or STAT3 genes in $20% of 155 ALK À ALCLs and demonstrated that 38% of systemic ALK À ALCLs displayed double lesions'' therefore should be ''88'' instead. The correct sentence should read as follows: ''We identified activating mutations of JAK1 and/or STAT3 genes in $20% of 88 ALK À ALCLs and demonstrated that 38% of systemic ALK À ALCLs displayed double lesions.'' The error has been corrected in the online version of the article.

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing.

Introduction. If the role of ALK chimeras is well known in the pathogenesis of ALK+ Anaplastic Large Cell Lymphoma (ALCL), the mechanisms leading to the transformation of ALK- ALCL remain elusive. Combining Whole Exome Sequencing (WES), Copy Number Variation analysis and RNAseq, we provide a comprehensive characterization of different driving genetic alterations leading to the constitutive activation of STAT3 in ALK- ALCL. Methods. The discovery panel was composed of 23 ALCL (5 ALK+ and 18 ALK). The frequency of JAK/STAT3 mutations was investigated by Sanger and deep sequencing analyses in an independent panel of 158 ALCL (88 ALK-, 26 ALK+ ALCL and 44 cALCL) and 67 PTCL. RNAseq analysis was performed in a total of 23 ALCL (18 ALK- and 5 ALK+ ALCL). Functional tests were performed by transfection/transduction of different cells lines (STAT3 -/- MEF, HEK-293T, SUPM2 TTA and SUPM2 S3S). Validation studies were executed via Western Blot, and using cells viability analyses, Luciferase Assay, 2D and soft agar colony assays. Results. WES of paired normal/tumoral DNA identified a broad number of missense mutations (n=752) with 54 genes mutated in at least two different samples, without any preferential chromosomal distribution. We focused on recurrent activating mutations in JAK1 and STAT3, known to have relevant biological activity, demonstrating that 20% of nodal ALK- ALCL and cutaneous ALCL displayed mutations in either one of them and that 38% of JAK1 or STAT3 mutants showed convergent lesions, with a single case harboring L910P JAK1, G1097D/V JAK1 and Y640F STAT3 mutations. Missense STAT3 mutations occurred within the SH2 domain (Y640F, N647I, D661Y and A662V), meanwhile recurrent JAK1 mutants targeted the kinase domain (G1097D/V). The forced expression of JAK1 mutants in MEF cells led to the constitutive activation of pSTAT3, a phenotype enhanced by co-expression of JAK1 and STAT3 mutants and linked to colony formation. The cell growth of single and double mutant MEF was efficiently controlled in vitro by JAK1/2 and Hsp90 inhibitors (ruxolitinib and PUH71). Similarly, the treatment of mice bearing a JAK1 and STAT3 mutated ALK- ALCL Patient Derived Tumorgraft (PDT) efficiently controlled the lymphoma cell growth. Next we proved that the transcriptome of reconstituted JAK1 or STAT3 mutated STAT3 MEF -/- cells showed a hyperactive STAT3 signaling pathway and the up-regulation of multiple transcription factors. Among STAT3 associated genes, we discovered ATF3 and its known transcriptional regulated genes. The relationship between pSTAT3 and ATF3 was strengthened by a Gene Set enrichment Analysis and by their down-regulation in NPM-ALK cells after genomic or pharmacological knockdowns (KD) of ALK signaling. Moreover, we showed that the protein levels of pSTAT3 and ATF3 concordantly decreased in NPM-ALK Karpas 299 cells treated with an ALK inhibitor (CEP28122) or after IL-2 starvation of NK/T-cells . The analysis of a large cohort of Peripheral T-cell lymphoma confirmed the preferential expression of “bona fide” STAT3 genes in ALK- (30%) and in ALK+ ALCL; data confirmed by immunohistochemistry approach. Because JAK1 and/or STAT3 mutations did not recapitulate all pSTAT3+ ALK- ALCL, we performed a massive parallel RNA sequencing in search of other lesions in this pathway. This approach recurrent in-frame fusions combining transcription/repressor factors (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were identified. These chimera (NFkB2/ROS1, NFkB2/TYK2, NCOR2/ROS1, PABP4/TYK2) were proven to have both transcriptional and enzymatic activities, and to be oncogenic, a phenotype that could be abrogated by a novel ROS1 inhibitor (JNJ-ROS1i-A). Lastly, we proved that NFkB2-ROS1 rescued the phenotype of NPM-ALK cells after ALK signaling ablation but not after STAT3 knock-down. Conclusions. Our data demonstrate that a subset of ALK- ALCLs displays the constitutive activation of JAK/STAT3 pathway via multiple and alternative mechanisms (single and convergent somatic mutations and translocations), which could be significantly abrogated by specific inhibitors (i.e. JAK1/2 and ROS1). The central role of STAT3 and the possibility of using novel target strategies provide new avenues in the treatment of ALK- ALCL patients, as suggested by our preclinical ALCL PDT model. Disclosures Rosenwald: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties.