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The interleukin-3 receptor [alpha] subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP- ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies., Introduction CD123, or the interleukin-3 (IL-3) receptor (IL-3R) [alpha] chain, is a cell-surface marker associated with a variety of hematologic malignancies and is often enriched on tumor cells compared with [...]

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity [...]

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers (1), but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβg dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling., Numerous somatic mutations are present in less than 5% of cases across multiple tumor types. To extensively catalog mutations in these 'long-tail' genes (2) will require sequencing thousands of additional [...]

The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7, which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer (1) and T-cell acute lymphoblastic leukaemia (T-ALL) (2). In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL (3-5), validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun (6), Myc (7), cyclin E (8) and notch 1 (ref. 9), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth (10), overexpression of Jun, Myc or notch 1 can also induce programmed cell death (11). Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Mbc and/or notch 1. Here we show that the E3 ubiquitin ligase [SCF.sup.FBW7] (a SKP1-cullin-1-F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7-deficient T-ALL., MCL1 is frequently overexpressed in various leukaemias through mechanisms that are not fully understood (12). MCL1 is distinct from other BCL2 family members in its extremely unstable nature (13), which [...]

Oncogenic FLT3 kinase is a clinically validated target in acute myeloid leukemia (AML), and both multi-targeted and selective FLT3 inhibitors have been developed. Spleen tyrosine kinase (SYK) has been shown to be activated and increased in FLT3-ITD-positive AML patients, and has further been shown to be critical for transformation and maintenance of the leukemic clone in these patients. Further, over-expression of constitutively activated SYK causes resistance to highly selective FLT3 tyrosine kinase inhibitors (TKI). Up to now, the activity of the multi-targeted FLT3 inhibitor, midostaurin, against cells expressing activated SYK has not been explored in the context of leukemia, although SYK has been identified as a target of midostaurin in systemic mastocytosis. We compared the ability of midostaurin to inhibit activated SYK in mutant FLT3-positive AML cells with that of inhibitors displaying dual SYK/FLT3 inhibition, targeted SYK inhibition, and targeted FLT3 inhibition. Our findings suggest that dual FLT3/SYK inhibitors and FLT3-targeted drugs potently kill oncogenic FLT3-transformed cells, while SYK-targeted small molecule inhibition displays minimal activity. However, midostaurin and other dual FLT3/SYK inhibitors display superior anti-proliferative activity when compared to targeted FLT3 inhibitors, such as crenolanib and quizartinib, against cells co-expressing FLT3-ITD and constitutively activated SYK-TEL. Interestingly, additional SYK suppression potentiated the effects of dual FLT3/SYK inhibitors and targeted FLT3 inhibitors against FLT3-ITD-driven leukemia, both in the absence and presence of activated SYK. Taken together, our findings have important implications for the design of drug combination studies in mutant FLT3-positive patients and for the design of future generations of FLT3 inhibitors.

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALKF1174L mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALKF1174L/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALKF1174L/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor–like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100–1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.

There remains a need to identify new sensitive diagnostic and predictive blood-based platforms in lymphoma. We previously discovered a novel circulating microRNA (miRNA) signature in a Smurf2-deficient mouse model that spontaneously develops diffuse large B-cell lymphoma (DLBCL). Herein, we investigated this 10-miRNA signature (miR-15a, let-7c, let-7b, miR-27a, miR-10b, miR-18a, miR-497, miR-130a, miR24, and miR-155) in human lymphoma cell lines, mice engrafted with patient-derived xenografts (PDXs), and DLBCL patient serum samples leveraging systems biology analyses and droplet digital PCR (ddPCR) technology. Overall, 90% of the miRNAs were enriched in PDX DLBCL models and human lymphoma cell lines. Circulating miRNAs from the serum of 86 DLBCL patients were significantly increased compared with healthy controls and had similar patterns to the murine models. Strikingly, miRNAs were identified up to 27-fold higher levels in the serum of PDX-bearing mice and human patients compared with lymphoma cell lysates, suggesting a concentration of these factors over time within sera. Using cut-points from recursive partitioning analysis, we derived a 5-miRNA signature (let-7b, let-7c, miR-18a, miR-24, and miR-15a) with a classification rate of 91% for serum from patients with DLBCL versus normal controls. In addition, higher levels of circulating let-7b miRNA were associated with more advanced stage disease (i.e., III-IV vs. I-II) in DLBCL patients and higher levels of miR-27a and miR-24 were associated with MYC rearrangement. Taken together, circulating multi-miRNAs were readily detectable in pre-clinical cell line and human lymphoma models as well as in DLBCL patients where they appeared to distinguish clinico-pathologic subtypes and disease features. [ABSTRACT FROM AUTHOR]

The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Only25%of the DLBCLPDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition. [ABSTRACT FROM AUTHOR]

B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G2/M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer. [ABSTRACT FROM AUTHOR]