Search

Supplementary Figure 1 from The Tyrosine Phosphatase Shp2 Interacts with NPM-ALK and Regulates Anaplastic Lymphoma Cell Growth and Migration

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry–based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)–mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALKK210R, formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration. [Cancer Res 2007;67(9):4278–86]

/SummaryTo maintain blood homeostasis, millions of terminally differentiated effector cells are produced every day. At the apex of this massive and constant blood production lie hematopoietic stem cells (HSCs), a rare cell type harboring unique self-renewal and multipotent properties. A key feature of HSCs is their ability to temporarily exit the cell cycle in a state termed quiescence. Defective control of cell cycle progression can eventually lead to bone marrow failure or malignant transformation. It is thought that HSCs must re-enter the cell cycle in order to commit to terminal differentiation. However, the molecular mechanisms tying cell cycle re-entry to cell fate commitment in HSCs remain elusive. Here, we identify the chromatin-associated Sin3B protein as a molecular link between cell cycle progression and differentiation in HSCs. We demonstrate that Sin3B is necessary for HSCs’ commitment to differentiation, but dispensable for their self-renewal or survival. Single cell transcriptional profiling of hematopoietic stem and progenitor cells (HSPCs) inactivated for Sin3B reveals aberrant cell cycle gene expression, consistent with the observed aberrant progression through the G1phase of the cell cycle. The defective cell cycle control elicited upon Sin3B inactivation correlates with the engagement of discrete signaling programs, including aberrant expression of cell adhesion molecules and essential components of the interferon signaling cascade in LT-HSCs. Additionally, chromatin accessibility profiling in LT-HSCs reveals the Sin3B-dependent accessibility of genomic elements controlling HSC differentiation, suggesting a functional link between cell cycle progression, and priming of hematopoietic stem cells for differentiation. Together, these results point to controlled progression through the G1phase of the cell cycle as a likely regulator of HSC lineage commitment through the modulation of chromatin features.

Investigating how chromatin organization determines cell-type-specific gene expression remains challenging. Experimental methods for measuring three-dimensional chromatin organization, such as Hi-C, are costly and have technical limitations, restricting their broad application particularly in high-throughput genetic perturbations. We present C.Origami, a multimodal deep neural network that performs de novo prediction of cell-type-specific chromatin organization using DNA sequence and two cell-type-specific genomic features—CTCF binding and chromatin accessibility. C.Origami enables in silico experiments to examine the impact of genetic changes on chromatin interactions. We further developed an in silico genetic screening approach to assess how individual DNA elements may contribute to chromatin organization and to identify putative cell-type-specific trans-acting regulators that collectively determine chromatin architecture. Applying this approach to leukemia cells and normal T cells, we demonstrate that cell-type-specific in silico genetic screening, enabled by C.Origami, can be used to systematically discover novel chromatin regulation circuits in both normal and disease-related biological systems.

(223)Ra is a bone-seeking, α-particle–emitting radionuclide approved for the treatment of patients with metastatic prostate cancer and is currently being tested in a variety of clinical trials for primary and metastatic cancers to bone. Clinical evaluation of (223)Ra hematologic safety showed a significantly increased rate of neutropenia and thrombocytopenia in patients, hinting at myelosuppression as a side effect. Methods: In this study, we investigated the consequences of (223)Ra treatment on bone marrow biology by combining flow cytometry, single-cell RNA sequencing, three-dimensional multiphoton microscopy and bone marrow transplantation analyses. Results: (223)Ra accumulated in bones and induced zonal radiation damage confined to the bone interface, followed by replacement of the impaired areas with adipocyte infiltration, as monitored by 3-dimensional multiphoton microscopy ex vivo. Flow cytometry and single-cell transcriptomic analyses on bone marrow hematopoietic populations revealed transient, nonspecific (223)Ra-mediated cytotoxicity on resident populations, including stem, progenitor, and mature leukocytes. This toxicity was paralleled by a significant decrease in white blood cells and platelets in peripheral blood—an effect that was overcome within 40 d after treatment. (223)Ra exposure did not impair full hematopoietic reconstitution, suggesting that bone marrow function is not permanently hampered. Conclusion: Our results provide a comprehensive explanation of (223)Ra reversible effects on bone marrow cells and exclude long-term myelotoxicity, supporting safety for patients.

Chimeric antigen receptor (CAR) T cells in solid tumors have so far yielded limited results, in terms of therapeutic effects, as compared to the dramatic results observed for hematological malignancies. Many factors involve both the tumor cells and the microenvironment. The lack of specific target antigens and severe, potentially fatal, toxicities caused by on-target off-tumor toxicities constitute major hurdles. Furthermore, the tumor microenvironment is usually characterized by chronic inflammation, the presence of immunosuppressive molecules, and immune cells that can reduce CAR T cell efficacy and facilitate antigen escape. Nonetheless, solid tumors are under investigation as possible targets despite their complexity, which represents a significant challenge. In preclinical mouse models, CAR T cells are able to efficiently recognize and kill several tumor xenografts. Overall, in the next few years, there will be intensive research into optimizing novel cell therapies to improve their effector functions and keep untoward effects in check. In this review, we provide an update on the state-of-the-art CAR T cell therapies in solid tumors, focusing on the preclinical studies and preliminary clinical findings aimed at developing optimal strategies to reduce toxicity and improve efficacy.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and often incurable disease. To uncover therapeutic vulnerabilities, we first developed T-ALL patient–derived tumor xenografts (PDXs) and exposed PDX cells to a library of 433 clinical-stage compounds in vitro. We identified 39 broadly active drugs with antileukemia activity. Because endothelial cells (ECs) can alter drug responses in T-ALL, we developed an EC/T-ALL coculture system. We found that ECs provide protumorigenic signals and mitigate drug responses in T-ALL PDXs. Whereas ECs broadly rescued several compounds in most models, for some drugs the rescue was restricted to individual PDXs, suggesting unique crosstalk interactions and/or intrinsic tumor features. Mechanistically, cocultured T-ALL cells and ECs underwent bidirectional transcriptomic changes at the single-cell level, highlighting distinct “education signatures.” These changes were linked to bidirectional regulation of multiple pathways in T-ALL cells as well as in ECs. Remarkably, in vitro EC-educated T-ALL cells transcriptionally mirrored ex vivo splenic T-ALL at single-cell resolution. Last, 5 effective drugs from the 2 drug screenings were tested in vivo and shown to effectively delay tumor growth and dissemination thus prolonging overall survival. In sum, we developed a T-ALL/EC platform that elucidated leukemia-microenvironment interactions and identified effective compounds and therapeutic vulnerabilities.

Although the structure and function of the alphoid-tetO Human Artificial Chromosome (tetO-HAC) has been previously described in cell culture models and somatically in the mouse, in vivo persistence and stability throughout meiosis and across generations were not evaluated. Here we report germline transmission of a circular tetO-HAC across three mouse generations without observable health or reproductive deficiencies. Furthermore, we show that the tetO-HAC is maintained without selection as an episome and can be efficiently transmitted by both ova and sperm.

The mammalian genome is spatially organized in the nucleus to enable cell type-specific gene expression. Investigating how chromatin organization determines this specificity remains a challenge. Methods for measuring the 3D chromatin organization, such as Hi-C, are costly and bear strong technical limitations, restricting their broad application particularly in high-throughput genetic perturbations. In this study, we present C.Origami, a deep neural network model that performsde novoprediction of cell type-specific chromatin organization. The C.Origami model enablesin silicoexperiments to examine the impact of genetic perturbations on chromatin interactions in cancer genomes and beyond. In addition, we propose anin silicogenetic screening framework that enables high-throughput identification of impactful genomic regions on 3D chromatin organization. We demonstrate that cell type-specificin silicogenetic perturbation and screening, enabled by C.Origami, can be used to systematically discover novel chromatin regulatory mechanisms in both normal and disease-related biological systems.

NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR–Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway. Biochemical and genetics studies identify CRL4AMBRA1 as the ubiquitin ligase that has a key role in regulating the stability of D-type cyclins during cell-cycle progression.

Pulmonary disease increases the risk of developing abdominal aortic aneurysms (AAA). However, the mechanism underlying the pathological dialogue between the lungs and aorta is undefined. Here, we find that inflicting acute lung injury (ALI) to mice doubles their incidence of AAA and accelerates macrophage-driven proteolytic damage of the aortic wall. ALI-induced HMGB1 leaks and is captured by arterial macrophages thereby altering their mitochondrial metabolism through RIPK3. RIPK3 promotes mitochondrial fission leading to elevated oxidative stress via DRP1. This triggers MMP12 to lyse arterial matrix, thereby stimulating AAA. Administration of recombinant HMGB1 to WT, but not Ripk3−/− mice, recapitulates ALI-induced proteolytic collapse of arterial architecture. Deletion of RIPK3 in myeloid cells, DRP1 or MMP12 suppression in ALI-inflicted mice repress arterial stress and brake MMP12 release by transmural macrophages thereby maintaining a strengthened arterial framework refractory to AAA. Our results establish an inter-organ circuitry that alerts arterial macrophages to regulate vascular remodeling., Lung damage increases abdominal aortic aneurysm (AAA) incidence, but the mechanism was unclear. Here, the authors show that injured lungs leak HMGB1, increasing RIPK3 expression in arterial macrophages that subsequently alters mitochondrial function, leading to MMP12 expression and AAA development.

Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer(1), but the mechanisms that regulate their turnover are still being debated(2,3). Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4(AMBRA1) (also known as CRL4(DCAF3)) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR–Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.

Background. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few innovative treatment options. This is also contributed by the lack of models capable of capturing the complexity of the tumor and its microenvironment. Aims. To identify patient-specific vulnerabilities and novel therapeutic strategies in T-ALL and interrogate the mechanisms of the crosstalk between leukemic and stromal elements. Methods. We established a drug-testing platform using patient-derived-tumor-xenografts (PDTX) and a mixed-culture approach using E4ORF1-transduced endothelial cells (ECs) (Seandel M et al, PNAS 2008) to overcome host-mediated chemoresistance. We performed functional experiments using total and single-cell RNA sequencing. Results. First, we established a battery of 22 T-ALL PDTX models that matched both phenotypically (immune-histochemistry, flow cytometry) and genotypically (TCR rearrangement, transcriptome) with the primary patients' samples. We then challenged these models (n=14 samples belonging to different PDTX and serial passages within each model) with a library of compounds (n=433) targeting redundant proteins (n=634). Unsupervised clustering and Principal Component Analysis (PCA) demonstrated two clusters of T-ALL samples based on differential drug susceptibility. We could at least partially correlate these differences to specific transcriptomic signatures predictive of drug response (Figure 1A). We then defined a group of pan-active compounds across all models (n=40), which we validated using an independent screening with/without ECs (Figure 1B). We found that ECs counteracted the activity of selected compounds (i.e. TSA, THZ1 and MLN2238). By PCA, we observed distinct response profiles based on different T-ALL models. We vectorized the EC-rescue and found that the direction was the same across all 3 models tested, indicating that it relied on similar mechanisms regardless of model identity. Based on the known role of IGF1-IGFR1 as a supportive EC-rescue axis (Medyouf H et al, J Exp Med 2011), we performed the same screening with/without recombinant IGFBP-7 (500 ng/mL), a decoy IGF1 molecule. Remarkably, IGFBP-7 completely or partially abrogated the EC-mediated rescue of selected compounds [enzastaurin (PKC-β inhibitor), SC144 (GP130 inhibitor), CHIR124 (Chk1 inhibitor) and YM155 (Survivin inhibitor)] (Figure 1B). Drugs not rescued by ECs (n=30) were considered positive hits and 5 of them (ruxolitinib, tofacitinib, panobinostat, bortezomib, irinotecan) ultimately proved to be effective in vivo in randomized pre-clinical trials either alone or in combination (Figure 1C). Our stepwise endothelial-leukemia platform led to the discovery of "public" and "private" vulnerabilities and the proof-of-principle of prediction-guided in vivo pre-clinical trials. We propose a list of compounds that could be readily translated into T-ALL clinical trials (Figure 1D). We finally proved the validity of our platform using other disease models (i.e. B and T-lymphoma PDTXs). Mechanistically, at single-cell resolution, in vitro interacting T-ALL cells and ECs underwent reciprocal transcriptome changes, with T-ALL shifting towards stemness/undifferentiation and ECs towards tumor-ECs (TECs) phenotypes. Furthermore, in vitro EC-educated T-ALL cells mimicked distinct T-ALL subsets of the leukemic spleen of corresponding PDTX mice (Figure 1E). Conclusions. These data demonstrate that our EC-T-ALL culture system simulates in vivo conditions, offering a robust platform to study drug response, leukemia-host interactions and cell plasticity. This approach will improve the pre-clinical predictability of novel drugs/combinations for T-ALL, as well as for other hematologic malignancies, and propel the development of patient-tailored treatments. Figure 1 Figure 1. Disclosures Melnick: Janssen Pharmaceuticals: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Epizyme: Consultancy; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Elemento: AstraZeneca: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Owkin: Consultancy, Other: Current equity holder; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Janssen: Research Funding. Chiaretti: amgen: Consultancy; pfizer: Consultancy; novartis: Consultancy; Incyte: Consultancy. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

Introduction: Peripheral T-cell lymphomas (PTCLs) include heterogeneous entities of rare and aggressive neoplasms. The improved understanding of the biological/molecular mechanisms driving T-cell transformation and tumor maintenance has powerfully propelled new therapeutic programs. However, despite this progress, PTCLs remain an unmet medical need. Recurrent aberrations and the deregulated activation of distinct signaling pathways have been mapped and linked to selective subtypes. The JAK/STAT signaling pathway's deregulated activation plays a pathogenetic role in PTCL, including ALCL subtypes. STATs regulate the differentiation/phenotype, survival and cell-growth, metabolism, and drug resistance of T-cell lymphomas as well as host immunosuppressive microenvironments. Although many drugs' discovery programs were launched, a plethora of compounds has failed. Methods: We have discovered heterobifunctional molecules by an iterative medicinal chemistry SAR campaign that potently and selectively degrade STAT3 in a proteasome-dependent manner. Conventional PTCL cell lines and Patient Derived Tumor Xenograft (PDTX) and/or derived cell lines (PDTX-CL), carrying either WT- or mutated-STAT3, were exposed to increasing amounts (50nM⇒5µM) of STAT3-degraders. Proteins and mRNA transcripts (2⇒144hrs) were assessed by deep-proteomics and paired-end RNA sequencing, combined with WB/flow cytometry and qRT-PCR. Cell-titer-glo, cell titer blue, Annexin-V and S-cell cycle analyses were used as readouts. Chromatin accessibility, STAT3 DNA binding, 3D chromosomal architecture reorganization and 5-hmC profiling were assessed by ATACseq, CHIPseq and Hi-C and H3K27ac Hi-CHIP and mass-spectrometry. Drug testing/discovery combinations (96-well-plate) were performed using a semi-automated flow-cytometry. A battery of PTCL PDTX models were tested in pre-clinical trials. Results: Treatment of ALK+ ALCL (SU-DHL1) led to the rapid (~6hrs.) and profound down-regulation of STAT3 followed by the loss of canonical STAT3-regulated proteins (SOCS3, MYC, Granzyme B, GAS1, and IL2RA), without appreciable changes for other STAT family members (STAT1, STAT5b). In vitro, cytoplasmic, nuclear, and mitochondrial STAT3 downregulation was maintained up to 144 hrs. Loss of STAT3 in ALK+/- ALCL and BIA-ALCL cells was associated with major transcriptional changes (7116-10615 and 15114 DEGs in ALK- and ALK+ ALCL, respectively), underscoring public/shared as well as private time-dependent signatures. Main down-regulated pathways included JAK-STAT, MAPK, NF-kB, PI3K, TGFb, and TNFa. Comparison of STAT3 shRNA (ALK+ ALCL) and STAT3 degrader (ALK-/ALK+ ALCL) signatures demonstrated a substantial and concordant gene modulation (24hrs) among all models with the highest overlaps between ALK+ ALCL (Figure 3). To identify direct STAT3 gene targets, we analyzed CHIPseq peaks and predicted bindings sites, demonstrating that canonical genes, i.e., SOCS3, Granzyme B, GAS1, IL2RA, STAT3, and CD30, were significantly downregulated. Conversely, CD58, CD274, and MCH-I/II were upregulated at late time points. By mapping the STAT3 binding sites in ALK+ and ALK- ALCL, we have identified 1077 and 2763 STAT3 peaks within promoter/5'-/3'- and distant intergenic regions, corresponding to both coding and non-coding genes. Therapeutically, in vitro treatments led to cell cycle arrest and profound growth inhibition, and over time increased cell death of PTCL cells, including ALCL. Accordingly, growth inhibition of ALCL xenograft and PDTX tumors was also achieved (Figure 2). To identify drugs that could synergize withSTAT3-degrader activity, we tested a compound library (40) targeting pro-tumorigenic PTCL pathways as well as FDA-approved compounds. Ongoing studies are in progress. Conclusion: We have discovered selective STAT3 degraders which control PTCL growth. STAT3 degraders are powerful tools to define the STAT3 pathogenetic mechanisms and dissect genes/pathways to be targeted for T-cell lymphoma eradication. These data provide additional rationale for testing STAT3 degraders in the clinic for the treatment of aggressive malignancies including PTCL/ALCL. Figure 1 Figure 1. Disclosures Yang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: Owkin: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Johnson and Johnson: Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; One Three Biotech: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Horwitz: Affimed: Research Funding; Aileron: Research Funding; ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.

Differences in three-dimensional (3D) chromatin architecture can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer–promoter interactions, impacting gene expression and leading to human disease. Here we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) by using primary human leukemia specimens and examine the dynamic responses of this architecture to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP–seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD ‘fusion’ event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small-molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia. Analysis of 3D chromatin architecture in T-ALL identifies differences in intra-TAD interactions and TAD boundary insulation. Inhibition of oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions.

A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell–containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34 + cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation.

Summary Ex vivo gene therapy based on CD34+ hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34+ cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34+CD38− cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing., Highlights • CD34+CD38− cells as an HSC-enriched starting population for ex vivo gene therapy • Reduced culture time (, In this article, Gentner and colleagues undertake a comprehensive strategy to advance ex vivo genetic engineering of HSCs for gene therapy. They experimentally define an optimal strategy to purify HSCs, which allows uncoupling long-term from short-term hematopoietic reconstitution, and implement ex vivo conditions that best preserve their biological properties applying novel transduction-enhancing compounds and pyrimidoindole derivatives to support HSC expansion.

Summary Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function., Graphical Abstract Highlights ► miR-126 is a novel regulator of the HSC quiescence/proliferation equilibrium ► Reduction in miR-126 induces an expansion of long-term HSC without exhaustion ► Constitutive miR-126 expression promotes HSC quiescence and progenitor proliferation ► miR-126 attenuates PI3K/AKT activation in response to cytokine stimulation, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway to promote HSC quiescence and progenitor proliferation.

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)–ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry–based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)–mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALKK210R, formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration. [Cancer Res 2007;67(9):4278–86]

Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. We identified the ubiquitin ligase Huwe1 as a crucial regulator of HSC function via its post-translational control of the oncoprotein N-myc (encoded by Mycn). We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid-fate specification in mice. Through the use of a fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc expression was upregulated in response to stress or following loss of Huwe1, which led to increased proliferation and stem-cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, which suggested that the attenuation of N-myc by Huwe1 is essential for reestablishing homeostasis following stress.

MicroRNAs are essential regulators of normal and malignant hematopoiesis. miRNAs are relevant for gene therapy, since they can be exploited to fine-tune the expression profile of vector constructs or to alter viral tropism (GentnerN Chiriaco et al, 2014; Escobar et al, 2014) and described the function of miR-126 in HSC where it regulates the balance between quiescence and self-renewal (Lechman et al, 2012). We here report a novel role for miR-126 in the induction and maintenance of high-grade B cell malignancies. By ectopically expressing miR-126 in transplanted BM cells, we observed that up to 60% of mice (n=71) developed B cell malignancies. LV insertion site (IS) analysis revealed that all tumors were monoclonal. We then tracked back leukemic clone to different hematopoietic lineages prospectively purified from the mice 2-6 months before disease onset. IS sharing between normal lineages and leukemic clone suggests stem or multipotent progenitor cell as origin for most tumors. Importantly, we show that miR-126 is the direct cause of genesis and maintenance of leukemia, since leukemogenesis is abolished when miRNA expression is inhibited by doxycycline (doxy) using a tetracycline-repressible miR-126 cassette, and established symptomatic leukemia completely regresses when miR-126 is switched off by doxy through induction of apoptosis. Transcriptional profiling indicated that miR-126 regulates multiple genes in p53 pathway both in murine blasts and in normal human CD34+ cells. Previous work suggested expression of miR-126 in acute lymphoblastic leukemia (ALL) and germinal center lymphoma. To further establish the relevance of miR-126 in human disease, we measured miR-126 expression in blasts from 16 adult patients with ALL. miR-126 was highly expressed in most studied ALL cases (Phil+: n=11, Phil-: n=5), at similar levels as CD34+ cells. We then down-regulated miR-126 in primary blasts from human B-ALL patients (n=5), and we observed increased apoptosis and impaired engraftment in xenograft models after primary and secondary transplantation (miR-126/KD: n=32 mice; Ctrl: n=37 mice), demonstrating the relevance of miR-126 in human B-ALL. In conclusion, we present a novel spontaneous mouse model for high grade B cell malignancies which are addicted to miR-126 expression, provide insight into the dynamic process of leukemogenesis by clonal IS tracking and unveil key tumor signaling pathways controlled by miR-126. Down-regulation of miR-126 could be exploited as therapeutic strategy in ALL, since it would deplete leukemic cells while expanding normal HSC, two ways to restore normal hematopoieis.

Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. To potentially improve the efficacy, safety and economic sustainability of HSPC transduction, we reasoned to genetically manipulate only the more potent CD34+CD38- HSPC, thereby improving HSPC maintenance in culture in the absence of differentiating cells and downscaling the cell therapy product by a factor of ten without compromising long-term engraftment. This approach would also decrease the total load of vector integration infused in the patients, thus improving its overall safety.First, we determined the engraftment kinetics of CD34+ mobilized peripheral blood (mPB) subpopulations over a 24wk xenotransplantation period. We sorted CD34+ mPB into 4 fractions with increasing expression levels of CD38 and marked each fraction with a specific fluorescent protein allowing to track the population of origin driving hematopoietic reconstitution. Differentially labeled fractions were mixed, and various combinations of CD38-, CD38int and CD38hi HSPC were injected into NSG mice (2 exp, n=30). Almost all long-term repopulating capacity (>90%) was contained within CD34+CD38- cells, and these cells took over hematopoiesis by 9wks. Instead, early reconstitution was mainly driven by CD34+CD38int progenitor cells.In the prospect of a clinical translation, we then modeled the co-administration of gene-modified CD34+CD38- mPB cells with uncultured CD34+CD38int/+ supporter cells aimed to drive fast hematopoietic recovery (3 exp, n=38). Repopulation by gene-modified CD34+CD38- cells was slower (15wks) and incomplete ( 5 wks and re-established long-term (24wks) gene marking up to 85%, thus allowing to benefit from prompt hematopoietic recovery driven by transiently repopulating CD38int/+ supporter cells.Last, we optimized LV transduction in the framework of an improved culture protocol. Exposing CD34+ or CD34+CD38- mPB cells to prostaglandin E2 (PGE2) increased transduction efficiency 1.5-2.5x, allowing to markedly reduce pre-stimulation and LV exposure times better preserving HSC functions. Importantly, the higher gene-transfer efficiency was maintained for up to 24 wks following xenotransplantation (n=33), suggesting that PGE2 facilitated LV transduction in long-term HSC.In summary, these results support the clinical development of novel HSPC gene therapy protocols based on the modification of highly purified HSC subsets, with the prospect to improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.

Anaplastic large cell lymphomas (ALCLs) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is frequently fused to the nucleophosmin (NPM) gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo and that ALK activity is strictly required for the survival of ALK-positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK-positive ALCL cell lines, abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell-permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPbeta and the antiapoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions.

In non-small cell lung cancer (NSCLC), receptor tyrosine kinases (RTKs) stand out among causal dominant oncogenes, and the ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nonetheless, long-term RTK inhibition leads invariably to acquired resistance, tumor recurrence and metastatic dissemination. In ALK+ cell lines, inhibition of ALK signaling was associated with coactivation of several RTKs, whose pharmacological suppression reverted the partial resistance to ALK blockade. Remarkably, ERBB2 signaling synergized with ALK and contributed to the neoplastic phenotype. Moreover, the engagement of wild-type epidermal growth factor receptor or MET receptors could sustain cell viability through early growth response 1 (EGR1) and/or Erk1/2; Akt activation and EGR1 overexpression prevented cell death induced by combined ALK/RTK inhibition. Membrane expression of ERBB2 in a subset of primary naive ALK+ NSCLC could be relevant in the clinical arena. Our data demonstrate that the neoplastic phenotype of ALK-driven NSCLC relays ‘ab initio' on the concomitant activation of multiple RTK signals via autocrine/paracrine regulatory loops. These findings suggest that molecular and functional signatures are required in de novo lung cancer patients for the design of efficacious and multi-targeted ‘patient-specific' therapies.

We have recently shown that miR-126 expression faithfully identifies the engrafting fraction of bone marrow (BM) and cord blood (Gentner et al, Sci Transl Med 2010), in which it regulates hematopoietic stem cell (HSC) pool size by modulating cell cycle progression (Lechman et al., Cell Stem Cell 2012). Antagonizing miR-126 expands HSC by enhancing PI3K/AKT signaling, without causing their exhaustion or transformation. By reconstituting mice with lentivirally transduced BM cells ectopically expressing miR-126, we noted differential effects in HSC and progenitors: while miR-126 overexpressing (126OE) HSC were more quiescent and outcompeted by HSC with physiologic miR-126 expression, progenitor subsets were increased in number and proliferated more upon 126OE. In particular, B lymphopoiesis was enhanced, with the appearance of 126OE-vector marked, CD45(low)CD19+Ig(-) B cell progenitors in the peripheral blood. This abnormal population was transiently observed in all 126OE (n=45), but in none of the control mice (n=30), and BM B cell precursors and progenitors from 126OE mice showed significantly reduced levels of apoptosis, as well as a differentiation block at the immature B cell stage. At least part of this effect was due to interference of miR-126 with p53 activation. Consequentially, 20-30% of 126OE mice (as compared to 0% of control mice) followed over 1 year developed vector-marked, high-grade B-cell neoplasms which resembled diffuse large B-cell lymphoma, lymphoblastic lymphoma or acute lymphoblastic leukemia (ALL). Strikingly, by using a tetracycline-regulated, conditional miR-126OE vector, we observed full regression of the expanded abnormal B cell population after switching miR-126 expression off by doxycycline administration, demonstrating that miR-126 is an oncogenic driver in hematologic malignancies. To establish the relevance of miR-126 in human disease, we studied primary samples from patients with acute leukemia. We measured miR-126 expression in blasts from 12 adult patients with acute lymphoblastic leukemia (ALL). miR-126 was highly expressed in all studied cases of ALL (Phil+: n=7, Phil-: n=5), often surpassing the levels found in normal HSC. Unlike in HSC, miR-126 expression was independent from expression of its host gene EGFL7, suggesting an ALL-specific regulation of the miR-126 locus. By exploiting a lentiviral miR-126 reporter vector, we quantified miR-126 activity with single cell resolution after xenotransplantation of miR-126 reporter vector transduced ALL blasts into NSG mice. In 7 out of 12 ALL cases, we identified distinct subpopulations exhibiting different levels of miR-126 activity, and miR-126 (hi) ALL cells were more frequently contained in the CD34+ cell fraction. Moreover, high miR-126 expression correlated with faster and higher ALL engraftment in NSG mice. In acute myeloid leukemia (AML), miR-126 is significantly enriched in leukemic stem cells (LSC) where it governs quiescence and chemotherapy resistance (see abstract by Lechman et al), and analogous studies in ALL are ongoing. To further substantiate the role of miR-126 in AML, we studied miR-126 activity in 11 primary AML cases (4 AML with CBF, core binding factor, mutations; 7 AML with normal karyotype) using the reporter vector technology and the NSG xenotransplantation model. Globally, we found highest miR-126 activity in CBF-mutated AML. However, there was substantial heterogeneity in subpopulations of each single disease. Significantly higher miR-126 levels were found in CD34+, and particularly in CD34+CD38- AML cells, a fraction that is enriched for LSC. To see whether there is a correlation between miR-126 expression and disease progression, we measured miR-126 levels in sorted blasts from paired diagnosis/relapse (n=6) and diagnosis/chemotherapy-refractory (n=8) patient samples. Strikingly, we found a significant up-regulation of miR-126 in the disease progression sample, suggesting a mechanistic role for miR-126 in AML persistence after chemotherapy. In summary, these data support a clinically relevant role of miR-126 in the pathogenesis, treatment response and progession of acute leukemias. Authors AG and SN contributed equally, and LN and BG contributed equally. Disclosures: No relevant conflicts of interest to declare.

Abstract 30 The precise mechanisms controlling hematopoietic stem cell (HSC) quiescence, self renewal and differentiation remain incompletely understood, and HSC expansion remains a long sought after goal for cell- and gene therapy. Genetic manipulation of single transcription factors and signal transduction pathways has so far failed to provide sustained HSC expansion, often resulting in stem cell exhaustion or the emergence of malignancy. MicroRNAs (miRNAs) post-transcriptionally regulate multiple genes in a coordinated fashion, making them attractive targets to manipulate signaling networks and complex cellular functions. We have systematically manipulated miR-126, a miRNA enriched in the primitive hematopoietic compartment, in mouse and human HSC by stably knocking down its activity with a sponge lentiviral vector (126/KD LV) or forcing its overexpression by a 126/OE LV. Hematochimeric mice were generated by transplanting 126/OE LV- or 126/KD LV- transduced progenitors into congenic mice (murine HSC) or NSG mice (human HSC), and the effects of altered miR-126 levels on hematopoiesis were investigated in vivo. Steady state hematopoiesis was comparatively normal, with no alterations in complete blood cell counts except a minor skewing towards or against lymphopoiesis upon 126/OE or 126/KD, respectively. Strikingly, under stress conditions such as 5-fluorouracil exposure, secondary transplantation or the NSG xenograft environment, 126/OE resulted in a progressive loss of HSC, while 126/KD increased HSC numbers as measured by immunophenotype and functional output upon competitive transplantation into secondary recipients. Limiting dilution analysis confirmed a 3–4 fold expansion of mouse and human HSC upon miR-126/KD, and the competitive advantage of 126/KD HSC was maintained long-term including post tertiary transplantation. To study the mechanism by which miR-126 regulates HSC numbers, cell cycle analysis was performed on 126/KD- or 126/OE progenitors isolated from steady state bone marrow. Human CD34+CD38-CD90+ and murine Lineage-Kit+Sca+CD150+ HSC were shifted towards the S/G2/M or the G0 phase upon 126/KD or 126/OE, respectively, indicating that miR-126 antagonism increased cycling while excessive levels of miR-126 favoured HSC quiescence within the niche microenvironment. Interestingly, this effect was highly specific for HSC and the most primitive progenitors. An in vitro culture model of HSC-enriched cord blood cells was set up to study the molecular basis of miR-126 mediated control of HSC cycling. This model reproduced key aspects of the HSC phenotype observed in vivo, i.e. increased or decreased proliferation of primitive progenitors upon 126/KD or 126/OE, respectively. Transcriptome analysis indicated that miR-126 manipulation altered several cellular functions and pathways, most significantly the PI3K/AKT/GSK3β signalling axis. We confirmed PIK3R2 and CRKII as direct miR-126 targets in CB CD34+38- cells and show enhanced PI3K pathway activation as reflected by increased AKT and GSK3β phosphorylation upon 126/KD, while 126/OE caused opposite effects. Pharmacologic inhibition of the beta subunit of PI3K abrogated the 126/KD phenotype, indicating that miR-126 mainly acts through the PI3K pathway. In contrast to published studies showing HSC expansion upon hyperactivation of the PI3K pathway using various genetic approaches, miR-126/KD did not cause HSC exhaustion or leukemogenesis. On the contrary, evidence will be provided that 126/OE is oncogenic in hematopoietic cells. Thus, miR-126 represents a principle target for HSC expansion for cell and gene therapy applications. In summary, we propose that in vivo HSC pool size is influenced by endogenous levels of miR-126 through modulation of the HSC quiescence/proliferation equilibrium. Disclosures: No relevant conflicts of interest to declare.

Abstract 2631 Little is known about microRNA function in hematopoietic stem and progenitor cells (HSPC). Using a lentivector genetic reporter strategy to functionally detect miRNA activity in hematopoietic cells at single cell resolution, we identified several miRNAs which were specifically expressed in mouse and human HSC and early progenitors, defined according to cell surface phenotype and functional repopulation assays. One of these HSPC-specific miRNAs, miR-126, was further studied. We generated a stable miR-126 knockdown (kd) or forced its expression (“knock-in”, ki) in mouse HSPC using lentiviral vectors. Kd or ki cells were competitively transplanted with congenic, control vector-transduced cells, and hematopoietic chimerism was followed for >1 year in both primary and secondary recipients. miR-126 kd HSPC displayed enhanced myeloid and/or lymphoid contribution during the early phases of reconstitution, while they subsequently contributed similarly as the control cells. When this steady state bone marrow (BM) was transplanted into secondary recipients, we noted an even more pronounced over-contribution of miR-126 kd cells to hematopoiesis. In the long run, however, some secondary mice showed signs of exhaustion of miR-126 kd cells. These data suggest that miR-126 kd enhances hematopoiesis, likely at the stem/early progenitor level and in particular under stress conditions. On the other hand, forced expression of miR-126 (ki) resulted in an early competitive disadvantage in vivo, with progressively decreasing contribution to all hematopoietic lineages, paralleled by a nearly complete depletion of Kit+Sca+Lin- (KSL) miR-126 ki cells in the BM at 6 weeks after transplant. At 3 weeks post-transplant, when miR-126 ki KSL cells could still be detected, we found an increased proliferative index in these cells as judged by EdU incorporation in vivo, paralleled by a higher hematopoietic output respect to control cells at week 2–4 after transplant. These data suggest that miR-126 ki might favor HSC commitment at the cost of self-renewal. This phenotype was specific for miR-126 and not due to vector toxicity, as we demonstrate stable, long term overexpression of several control miRNAs in vivo. Moreover, miR-126 ki cells showed normal clonogenic activity in vitro. We then optimized a protocol to stably knock down miR-126 in human cord blood (huCB) HSPC, and validated this approach by demonstrating upregulation of previously described miR-126 targets including the beta subunit of phosphoinositide-3-kinase. Manipulation of miR-126 activity changed cell growth and differentiation of huCB, and we show altered activation of key signal transduction pathways upon miR-126 kd. Identification of additional miR-126 targets is ongoing using unbiased proteomic and transcriptomic approaches. In summary, these data suggest that a narrow range of miR-126 activity is required for robust and sustained HSC function, and that its manipulation may provide novel insights into stem cell biology. Disclosures: No relevant conflicts of interest to declare.

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is the most common cancer in the world, and is lethal in 90% of the cases. In non-small cell lung cancer (NSCLC), deregulated receptor tyrosine kinases (RTKs) stand out among the causal dominant oncogenes. Consequently, the genomic and/or pharmacological ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nevertheless, this approach has serious constrains, probably due to the frequent co-activation of multiple RTKs in a considerable subsets of solid tumors. It has become also evident that during TK inhibitor-based regimens, RTK switching and/or the selection of resistant clones can frequently occur representing a serious and problematic drawback. In a subset of NSCLC, the Anaplastic Lymphoma Kinase (ALK) gene has been described to be translocated and fused to EML4. This determines the ALK constitutive dimerization and autophosphorylation leading to cellular transformation. Here, we investigated the ALK oncogenic addiction of human NSCLC and studied the putative co-operative role of other kinases. We first demonstrated that the ectopic expression of EML4-ALK in ALK positive NSCLC cell lines (H2228 and H3122) resulted in the activation of multiple signaling pathways, in manner similar to that described for other known ALK fusions. Although EML4-ALK can induce transformation in lung in vitro and in vivo, ALK inhibition via shRNA or small molecule inhibitors (CEPs, Cephalon) induced only the apoptosis of H3122 cells, whereas in H2228 it caused cell growth arrest. Moreover, the treatment with ALK inhibitors led to tumor regression, but not tumor eradication, in vivo. Based on Phosphoproteomic analyses we demonstrated that the phosphorylation status of several tyrosine kinases (such as, EGFR, Met, FGFR, Jak1 or IGFR) was affected by the ALK inhibition only in H2228 cell line, but not in H3122. Notably, the combined treatment with anti-ALK ([CEP14083][1], CEP2550, [CEP28122][2]) and -EGFR inhibitors resulted in an increased cell death of H2228 cells to values similar to those observed for ALK treated H3122 cells. Finally, gene expression analyses showed that known EGFR substrates were specifically down regulated upon the combined treatment in ALK positive H2228 cells. Our findings suggest that ALK signaling is required for the maintenance of the neoplastic phenotype of some ALK positive NSCLC cells and that its abrogation could represent a novel strategy for the treatment of a well-defined subset of human lung cancer (complete ALK addiction). More importantly, we showed that the tumor survival and maintenance of ALK positive neoplastic cells might relay on the concomitant activation of multiple RTKs. These findings further support the notion that molecular and functional signatures are required for designing molecular-based “patient specific” therapeutic protocols in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-309. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP14083&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom [2]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP28122&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom