Myeloproliferative neoplasms (MPNs) are clonal malignant disorders of hematopoiesis arising in the hematopoietic stem cell (HSC) compartment that are characterized by excessive production of mature blood cells of the myeloid lineage. Transformation to secondary acute myeloid leukemia (sAML) represents a significant cause of death among MPN patients and this transformation occurs mainly from the clone carrying the disease phenotype driver mutation. Current treatment options for MPN patients are not curative and are limited to symptomatic treatment. Therefore, identification of novel therapeutic approaches with a clear disease-modifying effect for the treatment of MPNs and intercepting their progression to sAML is an unmet medical need. Mutations in JAK2, thrombopoietin receptor (MPL), and calreticulin (CALR) are phenotypic drivers in the pathogenesis of MPN. CALR mutations (CALRmut) are the second most frequent in MPN. CALRmut are insertions or deletions resulting in a frameshift in the last exon of the gene, causing a loss of the KDEL ER-retention motif and generation of a 36 amino acid positively charged C-terminal neoantigen. Due to loss of the KDEL motif, CALRmut are not confined to the ER and through interaction with MPL are trafficked to the cell surface where they induce persistent MPL activation and oncogenicity. Immunotherapies engaging T cells, such as bispecific cluster of differentiation 3 (CD3) redirection antibodies, show promising response rates in the clinic. Here, we describe JNJ-88549968, a novel first in class T-cell redirecting bispecific antibody that selectively targets CALRmut with the potential to achieve cures by eliminating MPN clones. The mechanism of action of JNJ-88549968 is to act as a bridge between CALRmut MPN cancer cells and T cells, inducing T-cell activation with subsequent T-cell-mediated cytotoxicity to CALRmut cancer cells in vitro and in vivo. JNJ-88549968 recognizes CALRmut epitopes common to all known CALRmut types. Cell surface localization of CALRmut was confirmed in CD34 + cells from CALRmut MPN patients. In-depth characterization of T cells from CALRmut MPN patients, using CyTOF and functional assays, confirmed their fitness and functionality. These data validate T-cell redirection as a rational therapeutic strategy for MPN patients carrying CALRmut. JNJ-88549968 demonstrated selective binding to CALRmut cell lines and no measurable binding to CALR wild type cells. JNJ-88549968 led to CALRmut-selective T-cell activation and cytotoxicity to CALRmut-engineered cell lines in vitro. The activity of JNJ-88549968 was also explored in an autologous setting using CD34 + cells isolated from CALRmut MPN patients as target cells together with T cells isolated from the same patients. JNJ-88549968 elicited concentration-dependent cytotoxicity of patient-derived CALRmut CD34 + cells. JNJ-88549968-mediated cytotoxicity was observed against all tested CALRmut CD34 + cancer cells and was independent of the type of CALR mutation. Moreover, JNJ-88549968 mediated robust in vivo efficacy in two independent CALRmut-positive xenograft murine leukemia models. In an established disseminated model, treatment with JNJ-88549968 significantly increased lifespan (ILS) compared to vehicle-treated control mice. Secreted CALRmut protein can be found in CALRmut MPN patient plasma. Evaluation of the impact of soluble CALRmut on JNJ-88549968 activity, including CALRmut-patient derived whole blood studies, indicated no effect on the activity of JNJ-88549968 in vitro. Taken together, JNJ-88549968 is a novel first-in-class bispecific T-cell redirection antibody investigated for the treatment of CALRmut MPN. JNJ-88549968 is currently being advanced for clinical investigation in patients with MPN.

Although chemotherapy induces complete remission in the majority of acute myeloid leukemia (AML) patients, many face a relapse. This relapse is caused by survival of chemotherapy-resistant leukemia (stem) cells (measurable residual disease; MRD). Here, we demonstrate that the anthracycline doxorubicin epigenetically reprograms leukemia cells by inducing histone 3 lysine 27 (H3K27) and H3K4 tri-methylation. Within a doxorubicin-sensitive leukemia cell population, we identified a subpopulation of reversible anthracycline-tolerant cells (ATCs) with leukemic stem cell (LSC) features lacking doxorubicin-induced H3K27me3 or H3K4me3 upregulation. These ATCs have a distinct transcriptional landscape than the leukemia bulk and could be eradicated by KDM6 inhibition. In primary AML, reprogramming the transcriptional state by targeting KDM6 reduced MRD load and survival of LSCs residing within MRD, and enhanced chemotherapy response in vivo. Our results reveal plasticity of anthracycline resistance in AML cells and highlight the potential of transcriptional reprogramming by epigenetic-based therapeutics to target chemotherapy-resistant AML cells.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Up to 30% of PTCL lack distinctive features and are classified as PTCL, not otherwise specified (PTCL-NOS). To further improve our understanding of the genetic landscape and biology of PTCL-NOS, we perform RNA-sequencing of 18 cases and validate results in an independent cohort of 37 PTCL cases. We identify FYN-TRAF3IP2, KHDRBS1-LCK and SIN3A-FOXO1 as new in-frame fusion transcripts, with FYN-TRAF3IP2 as a recurrent fusion detected in 8 of 55 cases. Using ex vivo and in vivo experiments, we demonstrate that FYN-TRAF3IP2 and KHDRBS1-LCK activate signaling pathways downstream of the T cell receptor (TCR) complex and confer therapeutic vulnerability to clinically available drugs.

The polycomb repressive complex 2, with core components EZH2, SUZ12, and EED, is responsible for writing histone 3 lysine 27 trimethylation histone marks associated with gene repression. Analysis of sequence data from 419 T-cell acute lymphoblastic leukemia (T-ALL) cases demonstrated a significant association between SUZ12 and JAK3 mutations. Here we show that CRISPR/Cas9-mediated inactivation of Suz12 cooperates with mutant JAK3 to drive T-cell transformation and T-ALL development. Gene expression profiling integrated with ChIP-seq and ATAC-seq data established that inactivation of Suz12 led to increased PI3K/mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), and WNT signaling. Moreover, a drug screen revealed that JAK3/Suz12 mutant leukemia cells were more sensitive to histone deacetylase (HDAC)6 inhibition than JAK3 mutant leukemia cells. Among the broad genome and gene expression changes observed on Suz12 inactivation, our integrated analysis identified the PI3K/mTOR, VEGF/VEGF receptor, and HDAC6/HSP90 pathways as specific vulnerabilities in T-ALL cells with combined JAK3 and SUZ12 mutations.

Next-generation sequencing has provided a detailed overview of the various genomic lesions implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Typically, 10–20 protein-altering lesions are found in T-ALL cells at diagnosis. However, it is currently unclear in which order these mutations are acquired and in which progenitor cells this is initiated. To address these questions, we used targeted single-cell sequencing of total bone marrow cells and CD34 + CD38 − multipotent progenitor cells for four T-ALL cases. Hierarchical clustering detected a dominant leukemia cluster at diagnosis, accompanied by a few smaller clusters harboring only a fraction of the mutations. We developed a graph-based algorithm to determine the order of mutation acquisition. Two of the four patients had an early event in a known oncogene ( MED12 , STAT5B ) among various pre-leukemic events. Intermediate events included loss of 9p21 ( CDKN2A/B ) and acquisition of fusion genes, while NOTCH1 mutations were typically late events. Analysis of CD34 + CD38 − cells and myeloid progenitors revealed that in half of the cases somatic mutations were detectable in multipotent progenitor cells. We demonstrate that targeted single-cell sequencing can elucidate the order of mutation acquisition in T-ALL and that T-ALL development can start in a multipotent progenitor cell.

T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia.

Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential.