Cytokine-independent Proliferation Research Articles

Abstract 9: AFNT-212: A TRAC-knocked-in KRASG12D-specific TCR-T cell product enhanced with CD8αβ and a chimeric cytokine receptor for treatment of solid cancers

Abstract The KRASG12D mutation is an ideal target for anti-cancer therapies as its expression is typically clonal, restricted to cancer tissue, and is among the most common oncogenic drivers in solid tumors. TCR-T cell therapies have demonstrated clinical activity in some solid cancers but have been limited by heterogeneous antigen expression and unfavorable tumor microenvironments. By targeting the KRASG12D mutation for which the cancer has established genetic dependency, AFNT-212 is designed to selectively target all cancer cells while avoiding on-target/off-tumor toxicities. AFNT-212 is non-virally engineered to knock-in a 5-transgene cassette expressing a high-avidity TCR specific for the KRASG12D mutation, a CD8α/β coreceptor, and a chimeric cytokine receptor. Transgene insertion at the TRAC locus disrupts expression of the endogenous TCRα, further enhancing the expression/activity of the transgenic KRASG12D TCR. Primary human CD8+ and CD4+ T cells were genetically engineered by a novel CRISPR-Cas nuclease system to integrate AFNT-212 transgenes within the TRAC locus. A cGMP compatible scale-up process for non-viral knock-in was established to support AFNT-212 clinical manufacturing. The activity of AFNT-212 was assessed against a panel of human KRASG12D tumor cell lines in vitro and established mouse xenograft models in vivo. The preclinical safety profile of AFNT-212 was evaluated by X-scan and crossreactivity assessment, alloreactivity studies, and cytokine independent growth studies. The specificity of gene-editing (GE) was assessed by an unbiased oligo-capture method followed by targeted sequencing. AFNT-212 TCR-T cells demonstrated potent in vitro anti-tumor activity against endogenously expressing HLA-A*11:01 KRASG12D tumor cells, including during chronic exposure to viable tumor cells. AFNT-212 TCR-T cells showed robust antitumor activity in established xenograft mouse models in vivo. No cross-reactivity was identified for the KRASG12D TCR against potential self-peptides even at supraphysiological levels, demonstrating high specificity of the TCR. No alloreactivity or cytokine-independent proliferation was observed. GE safety evaluations did not reveal any off-target activity using high sensitivity (~0.1%) NGS-based analyses or any GE-associated chromosomal rearrangements. The manufacturing of AFNT-212 consistently delivered >50-fold expansion of engineered TCR-T cells to meet expected clinical dose levels and exhibit memory/stemness phenotypes and negligible markers of immunologic exhaustion. AFNT-212, a novel TCR T cell therapy targeting KRASG12D mutant tumors, demonstrates robust activity against KRASG12D mutant tumors in vitro and in vivo. The robust manufacturing process developed using non-viral gene editing in the TRAC locus will support future clinical development of AFNT-212. Citation Format: Allison Drain, Nicholas Rouillard, Nathaniel Swanson, Martina Canestraro, Santosh Narayan, Tyler Warner, Nicole Danek, Ken Gareau, Jinsheng Liang, Luhua Shen, Tanya Tetrault, Iqraa Priyata, Sarah Vidyasagar, Taylor Riggins-Walker, Hui-Wen Liu, Klaus Pechhold, Lauren Brown, Joshua Francis, Xingyue He, Patrick Browne, Rebecca Lamothe, Meghan Storlie, Gregory Cost, Thomas M. Schmitt, Philip D. Greenberg, Smita S. Chandran, Christopher A. Klebanoff, Hubert Lam, Ankit Gupta, Damien Hallet, Gary Shapiro, Kim Nguyen, Loïc Vincent. AFNT-212: A TRAC-knocked-in KRASG12D-specific TCR-T cell product enhanced with CD8αβ and a chimeric cytokine receptor for treatment of solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 9.

IDH2/R140Q mutation confers cytokine-independent proliferation of TF-1 cells by activating constitutive STAT3/5 phosphorylation

BackgroundR140Q mutation in isocitrate dehydrogenase 2 (IDH2) promotes leukemogenesis. Targeting IDH2/R140Q yields encouraging therapeutic effects in the clinical setting. However, therapeutic resistance occurs in 12% of IDH2/R140Q inhibitor treated patients. The IDH2/R140Q mutant converted TF-1 cells to proliferate in a cytokine-independent manner. This study investigated the signaling pathways involved in TF-1(R140Q) cell proliferation conversion as alternative therapeutic strategies to improve outcomes in patients with acute myeloid leukemia (AML) harboring IDH2/R140Q.MethodsThe effects of IDH2/R140Q mutation on TF-1 cell survival induced by GM-CSF withdrawal were evaluated using flow cytometry assay. The expression levels of apoptosis-related proteins, total or phosphorylated STAT3/5, ERK, and AKT in wild-type TF-1(WT) or TF-1(R140Q) cells under different conditions were evaluated using western blot analysis. Cell viability was tested using MTT assay. The mRNA expression levels of GM-CSF, IL-3, IL-6, G-CSF, leukemia inhibitory factor (LIF), oncostatin M (OSM), and IL-11 in TF-1(WT) and TF-1(R140Q) cells were quantified via RT-PCR. The secretion levels of GM-CSF, OSM, and LIF were determined using ELISA.ResultsOur results showed that STAT3 and STAT5 exhibited aberrant constitutive phosphorylation in TF-1(R140Q) cells compared with TF-1(WT) cells. Inhibition of STAT3/5 phosphorylation suppressed the cytokine-independent proliferation of TF-1(R140Q) cells. Moreover, the autocrine GM-CSF, LIF and OSM levels increased, which is consistent with constitutive STAT5/3 activation in TF-1(R140Q) cells, as compared with TF-1(WT) cells.ConclusionsThe autocrine cytokines, including GM-CSF, LIF, and OSM, contribute to constitutive STAT3/5 activation in TF-1(R140Q) cells, thereby modulating IDH2/R140Q-mediated malignant proliferation in TF-1 cells. Targeting STAT3/5 phosphorylation may be a novel strategy for the treatment of AML in patients harboring the IDH2/R140Q mutation.BWcAJaJC8rGH42opvAqQCZVideo

Functional Consequence and Therapeutic Targeting of Cryptic ALK Fusions (ALK fus) in Monosomy7 AML

Monosomy 7 (Mono7) AML is one of the most established adverse prognostic markers in AML with over four decades of data on response and outcome in varied clinical trials. However, despite intensive and myeloablative therapies, outcome for this subtype of AML remains dismal with no meaningful advances in therapy in decades. As part of our recently completed discovery efforts in childhood AML (TARGET Pediatric AML; TpAML), we interrogated the transcriptome of 1068 children and young adults treated on COG AAML1031, including 28 cases of Mono7. We discovered novel cryptic fusions involving the Anaplastic Tyrosine Kinase (ALK) gene that were exclusively seen in 4 of 28 Mono7 patients (14.3%) and not detected in the remaining 1064 patients (p<0.001). These ALK fusions (ALK fus) included SPTBN1-ALK (n=3) or RANBP2-ALK (n=1) (figure 1A) and all 4 cases included the entire kinase domain of the ALK gene. Expression of ALK transcript was evaluated in Mono7 patients with and without ALK fus to determine whether these fusions lead to induction/upregulation of ALK. As demonstrated in figure 1B, the only patients with any ALK expression were those with ALK fus positive Mono7.Expression of the resultant onco-proteins of the ALK fus was confirmed by transfection of parental HEK293T cells with the oncofusion and western blot analysis of the protein lysates. As shown in the western blot in figure 1C, immunoblotting of the lysates from ALK fus cells with ALK antibody confirmed expression of the appropriate ALK+ fusion oncoprotein. Under experimental conditions, fusion involving SPTBN1-ALK appeared to generate higher levels of the fusion onco-protein compared to that of RANBP2-ALK fusion. Confirmation of the fusion onco-protein expression further validates the possibility of the functional significance and potential targetability of these fusions.The ALK gene codes for a receptor tyrosine kinase belonging to a family of protein kinases linked to unregulated cell growth which play a key role in CNS development. Alterations of the ALK gene are commonly seen in Non-Small Cell Lung Cancer (NSCLC), Anaplastic Large-Cell Lymphomas, and Neuroblastomas. We further inquired about the functionality of the ALK fus and whether they may confer cytokine-independent proliferation. The ALK fus were cloned from patient samples into pCSII, a direct GFP-tagged lentiviral backbone under the Ef1α promoter; followed by Sanger sequencing to verify the insert. IL-3 dependent Ba/F3 cells were transduced with the GFP tagged lentiviral vectors containing the SPTBN1-ALK or RANBP2-ALK fusion transcripts.Transduced cells were sorted to GFP homogeneity and growth parameters evaluated post-IL-3 withdrawal. In contrast to the parental line, which rapidly died in the absence of cytokines, cells expressing either SPTBN1-ALK or RANBP2-ALK sustained growth and rapidly proliferated in cytokine-free media, suggesting a conferred transformation event by the fusions, figure 1D. SPTBN1-ALK fusion appears to show more transforming potential with more rapid cytokine-free proliferation compared to that of RANBP2-ALK fusion. The observed differential growth pattern with the two ALK fus correlates with the differential expression of the two oncoproteins as presented above.We then sought to evaluate the efficacy of Crizotinib as a potential therapy for aberrant ALK fus positive Mono7 AML. Crizotinib has been FDA-approved and demonstrated efficacy in treating ALK positive NSCLC. ALK fus -positive cells were treated with varying doses of Crizotinib in cytokine-free media and cell viability and cell death were determined with the Promega Cell Titer Glow assay after 48 hours in culture. Fusion positive cells show sensitivity to Crizotinib with IC50s of 144nM and 95nM for the SPTBN1-ALK or RANBP2-ALK fusions, respectively.Here we demonstrate experimental data that when newly discovered cryptic ALK fus in Mono7 AML are functional (translation to fusion onco-protein, conferring cytokine independence) and show susceptibility to the ALK inhibitor crizotinib. This data may support effective therapeutic targeting of a subset of this highly refractory AML. [Display omitted] DisclosuresHylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. OffLabel Disclosure:Crizotinib is a first-generation ALK inhibitor that is FDA-approved for the treatment of Non-Small Cell Lung Cancer, Anaplastic Lymphoma, and Neuroblastoma.

Oncogenic Cooperation Between IL7R-JAK-STAT Pathway Mutations.

Oncogenic Cooperation Between IL7R-JAK-STAT Pathway Mutations.

Functional Properties of Exon 17 KIT Mutations in Pediatric CBF AML Predict for Inferior Outcome and Enhanced Response to Tyrosine Kinase Inhibition: A Report from the Children's Oncology Group

Introduction: KIT mutations are detected in approximately 20-30% of core binding factor (CBF, t(8;21) and inv(16)/t(16;16)) AML and have been associated with outcome and response to tyrosine kinase inhibitors (TKI). Mutations tend to localize to 2 regions- the receptor's kinase activation loop in exon 17 (E17) and a region of the extracellular domain in exon 8 (E8). The prognostic significance of the 2 mutations has varied in previous series. In this analysis, the functional significance of E8) and E17 KIT mutations in vitro and their response to TKIs were evaluated. We hypothesized that activating variants might be associated with inferior clinical outcome and be more susceptible to KIT-targeted agents.Methods: COG AAML0531 enrolled 1,070 patients, of which 800 underwent targeted next generation sequencing of the entire KIT gene coding sequence to identify somatic KIT mutations. To evaluate the functional impact of the most common KIT mutations, E8 (D419G, D419indel) and E17 (D816V, N822K) mutations were engineered and transduced into HEK293 cells for evaluation of KIT phosphorylation (pKIT) by immunoblotting and into Ba/F3 cells for cytokine independent proliferation studies. Mutations that resulted in pKIT were evaluated for response to the TKIs dasatinib and crenolanib. We further correlated our mutational and functional analysis to clinical outcome data from COG AAML0531. We analyzed outcomes by treatment arms (no-gemtuzumab ozogamicin (no-GO) vs. GO), focusing on the GO arm given the potentially favorable impact of GO on CBF AML response.Results: A total of 247 CBF patients were enrolled, with 218 undergoing screening for somatic KIT mutations. Mutations were detected in 55 (25%) patients; 27 (49%) in E8, 26 (47%) in E17, and 2 (4%) involved both exons. Among the 109 patients with CBF AML treated on no-GO arm, KIT mutations were detected in 29/109 (27%); 14 (48%) in E8, 13 (45%) in E17, and 2 (7%) involved both exons. E8 mutations were a combination of missense mutations or 3-9 bp indels that maintained reading frame, while E17 variants were primarily missense mutations involving the D816 or N822 codons, while. Cells transduced with E8 mutations showed baseline receptor phosphorylation and no IL-3 independent proliferation. In contrast, E17 mutations showed autonomous receptor phosphorylation and IL-3 independent proliferation, with D816V showing more robust cellular proliferation and higher levels of pKIT (Fig1A).We further evaluated the cytotoxic effect of 2 TKIs in engineered cells with activating KIT mutations and KIT wild-type (WT). Neither parental line nor cells expressing KIT -WT had any response to the two TKIs with IC50s > 10,000μM, however cells transduced with the E17 variants D816V or N822K were exquisitely sensitive to the TKIs. Following exposure to dasatinib and crenolanib, N822K+ cells had an IC50 of 3.8nM and 28.3nM respectively, with D816V+ cells demonstrating similar IC50s of 13nM and 14.3nM respectively (Fig 1B).Given the differential activating potential of E8 vs. E17 variants, we evaluated the clinical implications of the mutations in the 2 regions among CBF patients. On the no-GO arm, E8 KIT mutations lacked clinical significance, with an overall survival (OS) of 81±20% vs. 80±9% (p=0.91) for patients with and without E8 mutations, with a corresponding disease-free survival (DFS) of 57±26% vs. 62±11% (p=0.75) and relapse risk (RR) of 43±28% vs. 36±11% (p=0.59) respectively. In contrast, activating E17 mutations were associated with adverse outcome with an OS of 56±27% vs. 85±7% for those with and without E17 mutations (p=0.02; Fig 1C), with a corresponding DFS 33±27% vs. 65±11% (p=0.05) and a RR of 67±30% vs. 32±11% (p=0.03) respectively. Interestingly, when we evaluated outcomes on the GO arm, patients with E17 mutations had an OS of 77±23% vs. 83±8% for KIT-WT (p=0.62), suggesting that GO may abrogate the adverse prognostic impact of E17 mutations.Conclusions: Somatic E17 KIT mutations lead to autonomous receptor phosphorylation and cellular proliferation and are associated with inferior clinical outcome when treated with conventional chemotherapy alone. This is in contrast to E8 mutations, which lack functional significance. KIT E17 mutations are uniquely sensitive to the cytotoxic effects of KIT inhibition. Consideration of TKI therapy for at least this subset of pediatric KIT + CBF AML is prudent given their inferior prognosis and this preclinical correlate. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacological Inhibition of Insulin Growth Factor-1 Receptor (IGF-1R) Alone or in Combination With Ruxolitinib Shows Therapeutic Efficacy in Preclinical Myeloproliferative Neoplasm Models.

Even after development of the JAK1/JAK2 inhibitor ruxolitinib, myeloproliferative neoplasm (MPN) patients require novel therapeutic options. While ruxolitinib can considerably improve quality of life and prolong survival, it does not modify the natural disease course in most patients. Moreover, resistance develops with prolonged use. Therefore, various combination treatments are currently being investigated. Published data provide a compelling rationale for the inhibition of insulin growth factor-1 receptor (IGF-1R) signaling in MPN. Here we report that genetic and pharmacological inhibition of IGF-1R selectively reduced Jak2V617F-driven cytokine-independent proliferation ex vivo. Two different structurally unrelated IGF-1R inhibitors ameliorated disease phenotype in a murine MPN model and significantly prolonged survival. Moreover, in mice, low-dose ruxolitinib synergized with IGF-1R inhibition to increase survival. Our data demonstrate preclinical efficacy of IGF-1R inhibition in a murine MPN model.

Genome wide CRISPR screening reveals a role for sialylation in the tumorigenesis and chemoresistance of acute myeloid leukemia cells.

Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1cells. More than 182 genes (p<0.01) were found to suppress the cytokine-independent growth of TF-1cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ-/-) mice injected with CMAS or SLC35A1-knockout TF-1cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy.

R-2-HG in AML… friend or foe?

R-2-HG in AML… friend or foe?

The JAK2 mutation.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders with overproduction of mature myeloid blood cells, including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). In 2005, several groups identified a single gain-of-function point mutation JAK2V617F in the majority of MPN patients. The JAK2V617F mutation confers cytokine independent proliferation to hematopoietic progenitor cells by constitutively activating canonical and non-canonical downstream pathways. In this chapter, we focus on (1) the regulation of JAK2, (2) the molecular mechanisms used by JAK2V617F to induce MPNs, (3) the factors that are involved in the phenotypic diversity in MPNs, and (4) the effects of JAK2V617F on hematopoietic stem cells (HSCs). The discovery of the JAK2V617F mutation led to a comprehensive understanding of MPN; however, the question still remains about how one mutation can give rise to three distinct disease entities. Various mechanisms have been proposed, including JAK2V617F allele burden, differential STAT signaling, and host genetic modifiers. In vivo modeling of JAK2V617F has dramatically enhanced the understanding of the pathophysiology of the disease and provided the pre-clinical platform. Interestingly, most of these models do not show an increased hematopoietic stem cell self-renewal and function compared to wildtype controls, raising the question of whether JAK2V617F alone is sufficient to give a clonal advantage in MPN patients. In addition, the advent of modern sequencing technologies has led to a broader understanding of the mutational landscape and detailed JAK2V617F clonal architecture in MPN patients.

Preclinical Evaluation of ALLO-605, an Allogeneic BCMA Turbocar TTM Cell Therapy for the Treatment of Multiple Myeloma

Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented efficacy in heavily pretreated relapsed and/or refractory multiple myeloma (MM) patients but may require further engineering to achieve their greatest potential. Enhancing cell expansion and persistence by providing cytokine support has been shown to improve the long-term antitumor activity of adoptively transferred CAR T cells in preclinical models. Combining CAR T cells with systemically-administered cytokines or cytokine mimetics can however result in toxicities and adverse events. Alternatively, cytokine signaling can be provided in a constitutive, CAR T cell-intrinsic fashion, by exogenous expression of a Constitutively Active Chimeric Cytokine Receptor (CACCR). CACCRs can be engineered by combining a membrane-tethered dimerization and JAK-binding domain derived from the thrombopoietin receptor (TpoR) fused to an intracellular signaling domain derived from a cytokine receptor. We investigated the impact of CACCR expression on the phenotype, functionality, persistence, and safety profile of allogeneic CAR T cells targeting BCMA to produce a second generation allogeneic BCMA "TurboCAR T™" candidate (ALLO-605). BCMA CAR T cells were generated from healthy donor T cells by lentiviral transduction with a CAR construct followed by genetic inactivation of the TRAC and CD52 loci using TALEN® gene editing. Allogeneic BCMA TurboCAR™ T cells, engineered for stoichiometric expression of the CAR and a CACCR via a self-cleaving peptide, were produced similarly. Constitutive expression of the CACCR during manufacturing had no negative effects on CAR T cell phenotype or yield and resulted in a product with over 60% stem cell memory/central memory T cells. In vitro, BCMA TurboCAR T™ cells showed enhanced cytokine secretion, polyfunctionality and improved serial killing activity. In a disseminated mouse model of multiple myeloma, BCMA TurboCAR T™ cells exhibited at least a 2-fold increase in peak expansion and enhanced survival and persistence compared to BCMA CAR T cells, resulting in prolonged antitumor responses and delaying relapses. Despite this enhanced persistence, we found that exposure to target cells was absolutely required for the expansion and long-term activity of BCMA TurboCAR T™ cells and no evidence of target- and cytokine-independent proliferation was observed. Since adoptive cell therapies have the potential to elicit toxicities in some patients, two alternative approaches to modulate BCMA TurboCAR T™ cell activity were investigated. First, we tested the ability of a CD20-based off-switch incorporated within the CAR to sensitize cells to rituximab and found effective depletion of BCMA TurboCAR T™ cells by complement or effector cells in vitro and in vivo in the presence of the antibody. In addition, we confirmed rapid inhibition of BCMA TurboCAR T™ cells by the protein tyrosine kinase inhibitor dasatinib, which has been shown to interfere with LCK activity. The prolonged persistence and antitumor responses seen in preclinical models along with a favorable safety profile of BCMA TurboCAR™ T cells support clinical investigation of ALLO-605 in relapsed or refractory multiple myeloma. Disclosures Sommer: Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Lin:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sutton:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Bentley:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Yoon:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Au:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Vargas-Inchaustegui:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Cheng:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Van Blarcom:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Panowski:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sasu:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

MPN-274: Mpl is Activated by Dimers of MPN-Linked Calreticulin Mutants Stabilized by Disulfide Bonds and Ionic Interactions

Myeloproliferative neoplasms (MPNs) are frequently driven by insertions and deletions within the gene encoding calreticulin (CRT) that alter the sequence and change the net charge of the C-terminal domain. CRTDel52 (type-I) and CRTIns5 (type-II) are recurrent mutations. Although oncogenic transformation requires both mutated CRT and the myeloproliferative leukemia protein (Mpl), the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. Our studies reveal that the novel C-terminal domain of CRTDel52 encodes specificity both for Mpl binding and for disulfide-mediated CRT dimerization. Disulfide-stabilized CRT dimers and multimers are observed in MPN patient-derived platelet lysates and in transfected mammalian cells. Cysteine mutations within both the C-terminal domain (C400A and C404A) and the N-terminal domain (C163A) of CRTDel52 diminish disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of crystalized CRT oligomers, we propose a structural model of the Mpl-activating CRTDel52 unit as a covalently linked dimer that is stabilized by disulfides and ionic interactions at both the C-terminal and N-terminal domains. In support of this model, we found that elimination of intermolecular disulfides and disruption of ionic interactions at the proposed dimerization interfaces are required to abrogate the ability of CRTDel52 to induce cytokine-independent proliferation of Ba/F3 cells via Mpl. Based on docking studies, we hypothesize that productive CRTDel52 dimers bind and activate Mpl dimers by inserting their C-terminal tails onto the ligand-binding pocket of Mpl and by engaging immature Mpl N-linked glycans via their conventional N-terminal glycan-binding site. Funding: This work was funded by NIH grant (R01 AI123957 to MR) and the University of Michigan Fast Forward Protein Folding Diseases Initiative. I.D.P. was supported by the NSF Division of Biological Infrastructure (award 1855425).

The combination of FLT3 and SYK kinase inhibitors is toxic to leukaemia cells with CBL mutations

Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine‐independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non‐receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3‐positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL‐positive leukaemia. Taken together, our results support the notion that mutant CBL‐expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.

Modulating Interlukin-7 (IL-7) cytokine receptor signaling to enhance the persistence and anti-tumor efficacy of Epstein-Barr Virus (EBV) specific T-cells in EBV positive malignancies

Abstract EBV-specific T-cells (EBVSTs) targeting viral proteins in EBV+ lymphoma patients have produced 50% clinical response. Limited T-cell expansion and persistence attributed to lack of cytokines in the tumor microenvironment (TME) remains a major challenge. IL-7 signaling has survival and anti-apoptotic effects on T-cells, however, IL-7 availability in vivo is limited and the IL7 receptor is downregulated on activated T-cells. We aim to overcome this limitation of suppressive TME by augmenting IL-7 signaling. We recently reported the transgenic expression of a constitutively active IL-7 receptor (C7R) to provide the proliferative and survival advantage in T-cells. Hence, we hypothesize that EBVSTs endowed with cytokine-independent C7R signaling will have resistance to the immunosuppressive TME and increased persistence and anti-tumor potency. C7R expressing EBVSTs demonstrated enhanced antigen-specific IFN-γ release and superior target specific cytotoxicity in-vitro. In contrast to unmodified EBVSTs, C7R-EBVSTs demonstrated EBV antigen dependent, cytokine-independent proliferation and survival, associated with increased phosphorylated STAT5. Furthermore, C7R signaling provided resistance against TGFβ, Arginase-I, and M2 macrophages, as demonstrated by increased proliferation attributed to decreased apoptosis in C7R-EBVSTs. Following adoptive transfer in an EBV+ murine xenograft model, C7R-EBVSTs persisted significantly longer and demonstrated tumor clearance, while unmodified EBVSTs failed. C7R signaling augments EBVSTs potency and persistence against suppressive components of TME in our preclinical models. Once effective, the findings will be translated to clinical trials in EBV+ malignancies.

The marrow stem cell niche in normal and malignant hematopoiesis.

The hematopoietic niche is composed of endothelial cells, mesenchymal stromal cells of several types, and megakaryocytes, and functions to support the survival, proliferation, and differentiation of normal hematopoietic stem cells (HSCs). An abundance of evidence from a range of hematological malignancies supports the concept that the niche also participates in the pathogenesis of malignant hematopoiesis, differentially supporting malignant stem or progenitor cells over that of normal blood cell development. In 2005, patients with myeloproliferative neoplasms were reported to harbor an acquired, activating, missense V617F mutation of the cytokine-signaling Janus kinase (JAK)-2, JAK2V617F , present in virtually all patients with polycythemia vera and half of patients with essential thrombocythemia and primary myelofibrosis. Using both in vitro and in vivo methods, several investigators have shown that in addition to driving cytokine-independent proliferation in HSCs, JAK2V617F contributes to these neoplasms by altering the hematopoietic niche. The role of both endothelial cells and megakaryocytes bearing JAK2V617F will be presented, which involves altering cytokine production within the niche, resulting in their differential support of mutant kinase-bearing stem cells over their normal counterparts, and imparting relative radiation resistance to stem cells. The clinical correlates of these findings will be discussed, as will their therapeutic implications.

Different Classes of ABL1 Fusions Activate Different Downstream Signalling Nodes

Background: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk subtype of ALL driven by a range of tyrosine kinase and cytokine receptor rearrangements. ABL1-class rearrangements (ABL1, ABL2, CSF1R and PDGFRB) account for 17% of Ph-like ALL cases in children, and are clinically important to identify as they can be therapeutically targeted with tyrosine kinase inhibitors (TKIs). While the p190 BCR-ABL1 fusion is well described, less is known about the function and downstream signalling by rare ABL1 fusions. We identified a rare ABL1 fusion, SFPQ-ABL1, in a paediatric B-ALL patient using RNA-sequencing. This fusion lacks the ABL1 Src-homology-3 (SH3) and part of the SH2 domain, which are retained in BCR-ABL1. Other ABL1 fusions, RCSD1-ABL1 and SNX2-ABL1, have a similar structure. In this work we have utilised phosphoproteomics and Stable Isotope Labelling by Amino Acids in Cell Culture (SILAC), as well as in vitro and in vivo models, to determine differential signalling pathways between SFPQ-ABL1 and BCR-ABL1.Methods: We cloned SFPQ-ABL1 from patient cDNA, and engineered SFPQ-ABL1 and BCR-ABL1 fusions to include or delete the SH2 and SH3 domains. We performed proliferation and viability assays to assess the ability of these fusions to transform Ba/F3 cells and test sensitivity to TKIs. We performed total phosphopeptide and phosphotyrosine enrichments and utilised mass spectrometry to identify the phosphoproteome activated by canonical SFPQ-ABL1 and BCR-ABL1. Over representation analysis was performed on phosphopeptides significantly differing between BCR-ABL and SFPQ-ABL (Log fold change cut-off > 2.5) using the Gene Ontology (GO) knowledge base under the biological process category. Furthermore, we compared the phosphoproteome of canonical SFPQ-ABL1 to SFPQ-ABL1 with the SH2 and SH3 domains reintroduced (SFPQ-ABL1+SH). We have also developed novel mouse models, using syngeneic transplantation, of SFPQ-ABL1 and SNX2-ABL1 driven leukaemia.Results: SFPQ-ABL1 expressing Ba/F3 cells are sensitive to cell death induced by TKIs that block ABL1. Interestingly, while SFPQ-ABL1 and BCR-ABL1 both effectively blocked apoptosis, SFPQ-ABL1 was less able to drive cytokine-independent proliferation. Phosphoproteomic analysis showed that BCR-ABL1 and SFPQ-ABL1 differentially activate downstream signalling pathways, including SH-binding proteins. Hierarchical clustering of phosphopeptides quantified from cells expressing canonical BCR-ABL1, SFPQ-ABL1, and SFPQ-ABL1+SH, demonstrated that BCR-ABL1 and SFPQ-ABL1+SH were more similar to each other than to SFPQ-ABL1. SFPQ-ABL1 expression resulted in phosphorylation of proteins involved in RNA processing, metabolism and splicing, suggesting that SFPQ region of SFPQ-ABL1 also contributes to signalling.Conclusions: In this study, we have utilised phosphoproteomics for the unbiased identification of signalling nodes that are required for the function of different classes of ABL fusions. We have developed novel in vitro and in vivo models to further understand how these fusions function to drive leukaemia. Our data also suggests that ABL1 fusion partners play a role beyond dimerization and transphosphorylation of the kinase domains in oncogenic signalling, but further study is needed to establish the contribution to leukaemogenesis. Establishing signalling pathways that are critical to the function of rare ABL1 fusions may inform clinical approaches to treating this disease. DisclosuresNo relevant conflicts of interest to declare.

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants

AbstractMutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a −1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3′-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.

Novel Mechanisms of Growth Inhibition By Histone Deacetylase Inhibitors in MPN

Histone deacetylase inhibitors (HDACi) are being evaluated in several clinical trials for the treatment of hematological malignancies and 2 of them have been approved for the treatment of T cell lymphoma. We have investigated the effectiveness of using 2 HDACi - Vorinostat (SAHA) and Panobionstat for treatment of systemic mastocytosis (SM) for which no treatments are available. SAHA and Panobinostat showed concentration dependent inhibition of proliferation in mastocytosis patient derived cell lines, HMC1.1 and HMC1.2 bearing either single mutation in the juxtamembrane domain KITG560V or double mutation also including the activation loop KITG560V,D816V, respectively. Interestingly HMC1.2 cells with 2 mutations in KITG560V, D816V were more sensitive to treatment with both SAHA (IC50 ~ 260 nM) and Panobinostat (IC50 ~11 nM) as compared to HMC1.1 cells (IC50 ~ 600 nM for SAHA and IC50 ~ 23 nM with Panobionstat) that have a single mutation in KIT. The inhibition of proliferation by HDACi treatment appeared to be associated with modulation of cell cycle and differentiation rather than induction of apoptosis. We further tested the effect of combining HDACi treatment with tyrosine kinase inhibitor, Dasatinib. Combination of Dasatinib (5 - 20 nM) with either SAHA or Panbionostat was more effective than any of the single drugs in HMC1.1 cells. However such a combination effect was not seen in HMC1.2 cells. Over 90% patients with aggressive systemic mastocytosis carry KITD816V mutation. This particular mutation of KIT is highly resistant to therapeutics and is associated with poor survival. Thus patients carrying the KITD816V mutation are likely to benefit from treatment with HDACi while juxtamembrane mutation bearing patients would benefit more from a combination of HDACi with kinase inhibitors such as Dasatinib. To further understand the mechanism behind HDACi inhibition of KITD816V mediated oncogenic signals, we expressed KITD814V (murine equivalent of human D816V) in myeloid cells and treated them with SAHA and Panobinostat. Cells expressing KITD814V showed cytokine independent proliferation and interestingly, cytokine independent proliferation was more sensitive to inhibition by both SAHA and Panbinostat as compared to the cytokine dependent proliferation. In contrast to HMC cells, inhibition of cytokine independent proliferation by HDACi in KITD814V oncogene expressing cells was associated with significant increase in apoptosis. The percent cells negative for Annexin V stain decreased from 74% in control to ~46% in 24h upon treatment with 500 nM SAHA or 10 nM Panbinostat (p< 0.001; n=3). Induction of apoptosis by HDACi in KITD814V oncogene expressing cells was significantly suppressed in presence of cytokine. Consistent with the in vitro data, treatment with SAHA prolonged the survival of mice transplanted with myeloid cells expressing KITD814V mutation as compared to the vehicle treated group (Median survival - 21d (veh), 34d (SAHA), p=0.0015, n=5). In addition to increased histone acetylation, treatment of cells with HDACi also leads to increase in acetylation of chaperone proteins such as HSP90 which can mediate proteasomal degradation of oncogenes and has been suggested as one of the mechanisms by which HDACi mediates growth inhibition. In contrast, we found no significant decrease in mutant KIT protein expression or AKT expression and phosphorylation upon treatment with either of the HDACi. Though expression of Pim1 a pro survival serine/threonine kinase was down-regulated by SAHA and Panobinostat. Treatment with HDACi was also associated with differential modulation of STAT3, STAT5 and ERK pathways. Treatment with both SAHA and Panobinostat down-regulated STAT5 phosphorylation and up-regulated ERK1/2 phosphorylation with more increase in ERK1/2 phosphorylation observed in cells with higher sensitivity to inhibition by HDACi. Though increased activation of the ERK pathway has been associated with leukemogenesis; there is some evidence to indicate that hyper activation of ERK pathway in absence of additional leukemogenic signals activates a pro senescence program. Differential expression of Pim1 and along with differential modulation of STAT and ERK pathways may contribute to differential sensitivity of the cells to HDACi and are likely to be of therapeutic value in aggressive systemic mastocytosis that has so far been associated with poor prognosis. DisclosuresNo relevant conflicts of interest to declare.

Role of LIM Kinase in Oncogenic Signaling from FLT3 and KIT Receptors and Its Targeting in Myeloid Leukemia

Acute Myeloid leukemia (AML) has a poor prognosis in part due to lack of elimination of leukemic cells and acquisition of drug resistance. In particular mutations in the activation loop of KIT and internal tandem duplication (ITD) in Flt3 receptor are associated with poor prognosis in AML. Interestingly, more than 90% patients with aggressive systemic mastocytosis (ASM) carry the KITD816V mutation which is highly resistant to conventional therapeutics and invariably associated with poor survival. Since the efforts to target the kinase activity of FLT3 and KIT have met with little success; the focus has shifted to targeting critical downstream signaling regulators. We have elucidated the role of a novel downstream regulator of FLT3-ITD and KITD816V mediated leukemic transformation - LIM kinase and evaluated the efficacy of a novel inhibitor of LIM kinase (LIMK), Liminib. LIMK, a serine/threonine kinase has 2 isoforms; LIMK1 and LIMK2. They are substrates of the Rho associated kinase (ROCK) which we have shown to play a critical role in leukemic transformation (Cancer Cell, 2011). They in turn act on cofilin and phosphorylation by LIMK inactivates cofilin and thereby modulates F-actin reorganization. Western blot analysis showed marked increase in phospho cofilin levels in cells expressing FLT3-ITD or KITD814V oncogenes as compared to their WT counterparts. Dependence of cofilin phosphorylation on activation of ROCK and LIMK was confirmed by treating the cells with an inhibitor of ROCK, H1152 and Liminib, inhibitor of LIMK. Both the inhibitors effectively reduced oncogene-induced phospho-cofilin levels. Liminib, the LIMK inhibitor also inhibited cytokine independent proliferation driven by FLT3-ITD or KITD814V (>90% at 100 nM). It also showed concentration dependent inhibition of proliferation in mutant oncogene bearing patient derived human cell lines such as MV411, Kasumi and HMC. FLT3-ITD expressing leukemic cells become resistant to Flt3 kinase inhibitor AC220 by acquiring additional point mutations. Interestingly, the cells expressing AC220 resistant FLT3-ITDD835F or FLT3-ITDF691L mutation showed similar sensitivity to Liminib as cells with FLT3-ITD. The growth inhibitory effect of Liminib was also validated in primary blasts from AML and SM patients. To further elucidate the mechanism of growth inhibition by Liminib in leukemic cells, we assayed for apoptosis induction in the treated cells. Cells expressing FLT3-ITD were significantly more susceptible (~ 73%) to induction of apoptosis as compared the WT-FLT3 cells (~33%) on treatment with Liminib. The extent of apoptosis induction by Liminib was similar in FLT3-ITD and AC220 resistant FLT3-ITDD835F or FLT3-ITDF691L mutation bearing cells. In addition to the effects of Liminib on F-actin polymerization; experimental evidence indicates involvement of mitochondrial pathway in the induction of apoptosis by Liminib. There was increase in accumulation of cofilin in the mitochondrial fraction in Liminib treated oncogene expressing cells as compared to the untreated cells. Phosphorylation of cofilin on Ser3 suppresses its mitochondrial translocation and treatment with LIMK inhibitor would inhibit cofilin phosphorylation leading to increased mitochondrial transport. Mitochondrial transport of cofilin has been observed with other inducers of apoptosis such as staurosporine and it possibly contributes to mitochondrial dysfunction during induction of apoptosis. To validate our pharmacologic findings using Liminib genetically and to assess which of the 2 isoforms of LIMK contributes to leukemogenesis, we generated LIMK1 and LIMK2 single and double knockout mice. 3 H-thymidine incorporation assay showed that deletion of either of the LIMK significantly reduced KIT D814V induced growth factor independent proliferation by approximately 50%. The extent of inhibition was more pronounced in Limk1-/- Limk2+/- or Limk1+/- Limk2-/- bone marrow cells (~80%) as compared to cells with deletion of a single isoforms of LIMK. Thus, there is some functional redundancy between the 2 isoforms and inhibition of both is likely to have better therapeutic value. Taken together we have identified a novel signaling pathway involving LIMK and cofilin that appear to be selectively activated in regulating oncogenic signaling in AML and MPN but not in normal hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Analysis of Linsitinib for the Treatment of Polycythemia Vera

Even after the discovery of JAK2V617F in majority of Polycythemia Vera (PV) patients and the generation of JAK2 inhibitors, the treatment of PV remains to be improved. An increased understanding of aberrant signaling in PV cells may yield novel drug targets for this patient population. The formation of erythroid colonies in the absence of exogenous erythropoietin (Epo), so called Epo-independent erythroid colonies (EECs), is a pathognomonic hallmark of PV. Over 20 years ago, Axelrad and colleagues proposed that EEC formation is not due to Epo-independence. Rather, they postulated that PV cells are hypersensitive to Insulin Growth Factor-1 (IGF-1). Subsequently, increased IGF-1 receptor (IGF-1R) phosphorylation was demonstrated in PV MNCs. In addition, serum concentrations of IGF-1 binding protein (IGF-1BP) are significantly elevated in PV patients. It has been shown that IGF-1 cooperates with JAK2V617F to increase JAK/STAT signaling, elevating constitutive phosphorylation of STAT factors and thereby augmenting aberrant proliferation. We therefore hypothesized that inhibition of IGF-1 signaling would ameliorate the PV phenotype, even in the presence of JAK2V617F.The dual IGF-1R and insulin receptor (IR) kinase inhibitor linsitinib (formerly called OSI-906) was used to investigate the contribution of the IGF-1 pathway to PV pathophysiology. In methylcellulose colony assays, linsitinib significantly reduced the EEC formation from PV MNCs. In contrast, in the presence of EPO, colony formation from either PV or healthy control MNCs was not affected. These data suggest that linsitinib specifically inhibits growth of the PV clone. Linsitinib likewise significantly reduced the cytokine independent proliferation of Ba/F3 cells expressing JAK2V617F but did not affect proliferation of Ba/F3 cells expressing either JAKWT or JAK2V617F in the presence of cytokine (mIL3). These data again argue that IGF-1 signalling is required for aberrant, cytokine independent growth. In addition to reducing proliferation, Linsitinib also induced apoptosis in IL3-independent JAK2V617F transduced Ba/F3 cells. Using the JAK2V617F bone marrow transplant (BMT) model, we investigated the effect of linsitinib on the PV phenotype in vivo. Linsitinib treatment significantly reduced the elevated RBC count, hematocrit and hemoglobin in this PV mouse model. In addition, the white blood cell count was decreased.The data presented here provide a pre-clinical rationale for expanding the investigation of linsitinib as well as similar IGF-1 pathway inhibitors for the treatment of PV. The effect of combining JAK2 and IGF-1 inhibition may prove especially promising in this patient population and is currently being investigated. DisclosuresNo relevant conflicts of interest to declare.

Deregulated JAK/STAT signalling in lymphomagenesis, and its implications for the development of new targeted therapies

Gene expression profiling has implicated several intracellular signalling cascades, including the JAK/STAT pathway, in the pathogenesis of particular subtypes of lymphoma. In marked contrast to the situation in patients with either acute lymphoblastic leukaemia or a myeloproliferative neoplasm, JAK2 coding sequence mutations are rare in lymphoma patients with an activated JAK/STAT “signature”. This is instead the consequence of mutational events that result in the increased expression of non-mutated JAK2; positively or negatively affect the activity of other components of the JAK/STAT pathway; or establish an autocrine signalling loop that drives JAK-mediated cytokine-independent proliferation. Here, we detail these genetic lesions, their functional consequences, and impact on patient outcome. In light of the approval of a JAK1/JAK2 inhibitor for the treatment of myelofibrosis, and preliminary studies evaluating the efficacy of other JAK inhibitors, the therapeutic potential of compounds that target JAK/STAT signalling in the treatment of patients with lymphoma is also discussed.