Primary Human AML Cells Research Articles

Targeting the membrane-proximal C2-set domain of CD33 for improved CAR T cell therapy

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between Tcell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) Tcells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33PAN antibodies). CD33PAN CAR Tcells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33neg leukemia. Compared to CD33V-set CAR Tcells, CD33PAN CAR Tcells showed greater invitro and invivo efficacy against several human AML cell lines withdiffering levels of CD33 without increased expression of exhaustion markers. CD33PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33PAN CAR Tcells had greater invitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR Tcells binding CD33 close to the cell membrane, providing the rationale to investigate CD33PAN CAR Tcells further toward possible clinical application.

NK-2 Homeobox Transcription Factor NKX2-3 Confers Differentiation Block in NPM1-Mutated in Acute Myeloid Leukaemia

Dysregulation of HOX homeobox family transcription factor (TF) gene expression is important in the pathogenesis of AML in particular in NPM1-mutated and MLL rearranged molecular subtypes. However, little is known about the role of NK2-homeobox (NKX2) family TF genes. NKX2 genes have critical roles in normal development of the central nervous system, thyroid, lung and pancreas. We observed high expression of NKX2-3 in patient AML samples and hypothesised the transcription factor might have a functional role in the disease. By qPCR, we found that NKX2-3 is highly expressed in normal HSCs but is rapidly down regulated as cells differentiate. In primary AML samples, it is highly expressed in 21-37% cases, including within the immunophenotypic LSC compartment, in particular in cases with NPM1 and/or FLT3-ITD mutations. Forced expression of NKX2-3 in normal murine KIT + bone marrow (BM) HSPCs transiently enhanced their clonogenic activity and impaired differentiation: NKX2-3-expressing HSPCs expressed higher levels of E2F or MYC target genes, and leukemia stem cell (LSC) maintenance genes. Congenic transplant experiments revealed that forced expression of NKX2-3 in normal BM HSPCs was sufficient to enhance multilineage engraftment across four months of follow up without inducing leukemia. In keeping with this, transplantation of Nkx2-3 -/- knockout BM cells led to significantly inferior engraftment compared with BM cells from litter mate control mice. Thus, while increased expression of Nkx2-3 enhances engraftment of normal stem cells in transplantation assays, loss of Nkx2-3 impairs it. Analysis of NKX2-3 high primary human AML cases demonstrated that HOXA9 was the most highly upregulated transcription factor gene compared with NKX2-3 low cases. We expressed both factors singly and together in murine KIT+ BM HSPCs: Hoxa9/NKX2-3 cells exhibited significantly higher clonogenic activity and reduced morphologic and immunophenotypic differentiation compared with Hoxa9/MTV cells. Congenic BM transplant experiments demonstrated that Hoxa9/NKX2-3 recipients developed leukemias significantly earlier than Hoxa9/MTV recipients (median 82 vs 155 days). BM morphology and immunophenotyping confirmed that Hoxa9/NKX2-3AMLs exhibited a greater degree of differentiation block than Hoxa9/MTV AMLs. Leukemia cells from Hoxa9/NKX2-3 recipients coordinately expressed higher levels of E2F or MYC target genes, and LSC maintenance genes. NKX2-3 KD to ~30% of control values reduced proliferation and induced morphologic and immunophenotypic differentiation in primary patient NPM1-mutated AML cells. NKX2-3 KD led to down regulation of E2F and MYC target genes and normal human CD34 + stem cell genes; and up regulation of myeloid differentiation genes. Using a CRISPR ribonucleofection approach to relocate NPM1c to the nucleus we confirmed that NKX2-3 expression in NPM1-mutant primary AML cells is sustained by mutant NPM1c. ChIPseq in NPM1-mutated primary patient and KO52 AML cells identified NKX2-3 binding peaks. As expected, DNA sequences under peak apices were strongly enriched for NKX2-3 binding motifs; there was also significant enrichment for RUNX and ETS family motifs, but not HOX/MEIS/PBX motifs. To identify genes potentially regulated by NKX2-3, genomic coordinates of strong binding peaks were mapped to the single nearest gene. Remarkably, among genes up regulated following NKX2-3 knockdown in primary human AML cells there was a highly significant enrichment of genes with putative regulatory elements bound by NKX2-3. Likewise, among genes down regulated in NKX2-3-expressing KIT+ murine HSPCs, there was a similar significant enrichment of genes with putative regulatory elements bound by NKX2-3. These data suggest that NKX2-3 functions as a transcription repressor at bound regulatory elements in primary AML cells. In summary, NKX2-3 is highly expressed in NPM1 & FLT3 mutated molecular subtypes of AML and serves to enhance self-renewal and the level of differentiation block through binding to and repressing regulatory elements of myeloid differentiation genes.

Abstract A37: PDZD2 is essential for steady-state hematopoiesis and its 37-kDa secreted product, sPDZD2, functions as a soluble tumor suppressor in AML

Abstract PDZ domain-containing protein 2 (PDZD2) is almost universally silenced through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein that it is cleaved into a 37kDa protein that is secreted to the tissue microenvironment (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using ELISA. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected lower levels in AMLs. In addition, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition, upregulation of differentiation markers (CD15/CD11b) and cell cycle arrest in G0/G1. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot. In vitro treatment with r-sPDZD2 led to inhibition of CREB(S133) phosphorylation, as well as increased GSK3b(S9), RSK1/2(S221/S227) and p53(S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a and CD7. To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts and hemoglobin levels, an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML. Citation Format: Marlise L Guerrero Schimpf, Courtney C Sparger, Hsuan-Ting Huang, Dean Wade, Maria E. Figueroa. PDZD2 is essential for steady-state hematopoiesis and its 37-kDa secreted product, sPDZD2, functions as a soluble tumor suppressor in AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A37.

PDZD2 Is Essential for Steady-State Hematopoiesis and Its 37-Kda Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP and erythroid progenitors, however, it is almost universally lost through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37kDa protein known as secreted PDZD2 (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using an ELISA-based detection method. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected significantly lower levels in AMLs (169 ± 17.53 vs 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines secreted significantly lower levels of sPDZD2 into the culture medium than HSPCs (28.71 ± 1.76 vs 4.62 ± 1.19 ng/mL; p<0.0001). Notably, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition (CellTiter-Glo, p<0.05 for all), differentiation (confirmed by upregulation of CD15/CD11b by flow cytometry) and cell cycle arrest in G0/G1. qRT-PCR analysis of cell cycle regulators in MV4-11 cells treated with r-sPDZD2 confirmed upregulation of CDKN2A (4.01-fold) and E2F4 (4.66-fold) and marked downregulation of multiple cyclin genes, including cyclins D and E, compatible with the cell cycle arrest observed by flow cytometry. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot for validation. In vitro treatment with r-sPDZD2 led to inhibition of ERK1/2 (T202/Y204, T185/Y187) and CREB (S133) phosphorylation, as well as decreased ß-Catenin expression and increased GSK3ß (S9), RSK1/2 (S221/S227) and p53 (S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a (39 vs 15%) and CD71 (58 vs 26%). To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts (9.04 ± 0.26 vs 7.58 ± 0.47 x106/µL) and hemoglobin levels (14.88 ± 0.46 vs 12.30 ± 0.81 g/dL) (p<0.05 for both), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML.

Abstract 1884: HFB9-2, a novel Galectin 9 neutralizing antibody for the treatment of AML and other cancers

Abstract Monoclonal antibodies targeting immune checkpoints have demonstrated clinical success in a range of tumor types; however sustained responses are achieved in a fraction of patients due to primary or secondary resistance to treatment. Recent evidence has implicated the pleiotropic immunosuppressive modulator Galactoside-binding lectin Galectin 9 (Gal-9) as a key factor in the tumor microenvironment that renders tumors resistant to current chemo and immunotherapies. High Gal-9 expression has been reported in different types of cancer, including hematological malignancies such as AML and ALL, and multiple solid tumors. Previously, we have described the characterization of an anti-human Gal-9 antibody, HFB9-2, with sub-nanomolar affinity for human, murine and cynomolgous Gal-9. HFB9-2 blocks the interaction of Gal-9 with its receptors TIM3 and CD44, leading to inhibition of Gal-9 mediated Th1 cell apoptosis and regulatory T cell expansion. HFB9-2 demonstrated significant anti-tumor efficacy and increased survival in a WEHI-164 syngeneic model as a single agent and in combination with anti-PD1 antibody. HFB9-2 is well tolerated in cynomolgous monkeys after administered by intravenous infusion as a single dose of 10, 50, 200 mg/kg, with no adverse effects and a NOAEL of 200 mg/kg. HFB9-2 developability profile assessment does not anticipate any stability and manufacturing issues. We hypothesize that targeting Gal-9 represents a valuable strategy to reduce immunosuppression and improve clinical response in selected cancer patients, in particular AML. Gal-9 has been reported to play a dual role in AML as both a self-renewal factor for leukemic stem cells and a suppressor of anti-cancer immunity. Analysis of AML patient serum samples demonstrated that Gal-9 expression was significantly higher than in healthy controls and that dropped at complete remission. Higher levels of Gal-9 were found in French-America-British (FAB) type M4 and M5 AML samples, and the lowest levels were observed in M3. To better understand the role for Gal-9 in the development of AML, we implanted primary human AML cells from different donors in M-NSG mice (NOD-Prkdcscid Il2rgem1). We demonstrate that the engraftment of primary human AML cells in these mice resulted in high levels of human Gal-9 in their serum that were detectable 2 weeks after intravenous implantation. The serum levels of Gal-9 increased over time following the increase of hCD45+ AML cells. Further analysis to evaluate the effect of Gal-9 neutralization in AML progression after primary and secondary engraftment with HFB9-2 are currently ongoing. The data presented here provide evidence that neutralization of Gal-9 with HFB9-2 blocks key immunosuppressive mechanisms known to favor cancer progression and to limit the efficacy of current immunotherapies, and together with the data obtained in AML patient samples position Gal-9 neutralization as a promising anticancer approach. Citation Format: Germain Margall, Muriel David, Julie Prigent, Dean Lee, Wenhua Xu, Joyce Pi, Xing Cai, He Zhou, Andreas Raue, Nicola Beltraminelli, Mingjie Chen, Jia Wu, Mingfang Feng, Angelo Paci, Julia Delahousse, Véronique Saada, Stéphane de Botton, Pierre Busson, Stephanie Beq, Francisco Adrian, Liang Schweizer, Yun-Yueh Lu. HFB9-2, a novel Galectin 9 neutralizing antibody for the treatment of AML and other cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1884.

Acute Myeloid Leukaemia Induces p16 Driven Senescence in the Bone Marrow Microenvironment to Support Their Proliferation and Survival

BackgroundAcute myeloid leukaemia (AML) occurs most commonly in older people and depends on the bone marrow microenvironment. The aging bone marrow accumulates senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of numerous pro-inflammatory cytokines, chemokines and growth factors, termed the secretary associated senescence profile (SASP). Previously we shown that AML is capable of reprogramming bone marrow stromal cells (BMSC) to support blast survival and proliferation. Here, we hypothesis that AML programs a senescent aging phenotype in the BMSC, generating a SASP in the BMSC which then feedback to promote AML blast proliferation in the bone marrow niche.MethodsWe isolated primary AML cells and BMSC from the bone marrow, with informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). We characterized BMSC by flow cytometry for expression of CD90+, CD73+, CD105+ and CD45, and assayed cytokines and chemokines secreted by primary AML cells, primary BMSC and AML/BMSC co-cultures using a proteomic array. To assess senescence of BMSC, we measured p16 mRNA and protein levels and senescence associated β-Galactosidase activity. We also transplanted syngeneic AML blasts (generated from lineage negative cells transduced with the oncogene MN1) into p16-3MR mice (Demaria, M., et al. 2014 Cell Development), in which the herpes simplex virus thymidine kinase (HSV-TK) moiety of the p16-3MR transgene allows selective killing of senescent p16-expressing cells by the pro-drug ganciclovir (GCV). We also used the NSG xenograft model in which we subcutaneously transplanted primary human AML cells and BMSC (either control and p16-kockdown).ResultsWe report that human AML cells induce BMSC to secrete a cytokine profile (including interleukin-6, interleukin-8, MIP1a and MIP2a). To examine whether AML cells can induce senescence in BMSC in culture, we co-cultured primary AML-derived BMSC with primary AML blasts and assayed for the senescent phenotype. We found that primary AML cells can induce p16 mRNA and protein and increase senescence associated β-Galactosidase activity in the BMSC after 6 days in culture. Furthermore, primary AML cells cultured on p16 knockdown BMSC had reduced survival compared to control wild type BMSC. In vivo we show that MN1 blasts engraft p16-3MR mice and the depletion of p16 positive stromal cells through treating animals with GVC compared to vehicle control significantly increased survival of MN1 engrafted mice. Finally, subcutaneous injection of human AML cells with human p16-KD BMSC significantly decreased tumour volume compared to injection of human AML cells with control-KD BMSC in mouse xenograft model.ConclusionHere we show that the AML blast induces a senescent phenotype in the bone marrow microenvironment which then feeds back to enhance the growth capacity of the AML blast. Targeting the senescent microenvironment may provide a novel therapeutic approach for the treatment of this disease. DisclosuresCampisi:Unity Biotechnology: Equity Ownership.

The PI3K/AKT Pathway Inhibitor ISC-4 Induces Apoptosis and Inhibits Growth of Leukemia in Preclinical Models of Acute Myeloid Leukemia.

Acute myeloid leukemia is a heterogeneous disease with a 5-year survival rate of 28.3%, and current treatment options constrained by dose-limiting toxicities. One of the key signaling pathways known to be frequently activated and dysregulated in AML is PI3K/AKT. Its dysregulation is associated with aggressive cell growth and drug resistance. We investigated the activity of Phenybutyl isoselenocyanate (ISC-4) in primary cells obtained from newly diagnosed AML patients, diverse AML cell lines, and normal cord blood cells. ISC-4 significantly inhibited survival and clonogenicity of primary human AML cells without affecting normal cells. We demonstrated that ISC-4-mediated p-Akt inhibition caused apoptosis in primary AML (CD34+) stem cells and enhanced efficacy of cytarabine. ISC-4 impeded leukemia progression with improved overall survival in a syngeneic C1498 mouse model with no obvious toxic effects on normal myelopoiesis. In U937 xenograft model, bone marrow cells exhibited significant reduction in human CD45+ cells in ISC-4 (~87%) or AraC (~89%) monotherapy groups compared to control. Notably, combination treatment suppressed the leukemic infiltration significantly higher than the single-drug treatments (~94%). Together, the present findings suggest that ISC-4 might be a promising agent for AML treatment.

Phase 1/1b Study of the Stapled Peptide ALRN-6924, a Dual Inhibitor of MDMX and MDM2, As Monotherapy or in Combination with Cytarabine for the Treatment of Relapsed/Refractory AML and Advanced MDS with TP53 Wild-Type

Background: ALRN-6924, the first-ever clinical stage stapled peptide, has been structurally stabilized (“stapled”) in an α-helical configuration, to mimic the inhibitor-binding region of the intracellular tumor suppressor protein, p53. By mimicking this region, ALRN-6924 binds the two most important endogenous inhibitors of p53, murine double minute-X (MDMX) and murine double minute-2 (MDM2), thereby restoring p53's ability to induce cell cycle arrest and apoptosis in TP53 wild-type (WT) cancer cells. ALRN-6924 is the first known synthetic agent to simultaneously target both of these important p53 inhibitors.In preclinical studies, ALRN-6924 inhibited the proliferation of AML cell lines and primary human AML cells alone and in combination with cytarabine. Preclinical data (Carvajal et al, 2018) demonstrated antiproliferative effects against leukemic stem cells, and complete responses that translated into cures in approximately 40% of mice in xenotransplantation studies. These data also suggested that more frequent dosing may enhance efficacy, and this hypothesis is now being evaluated in the ongoing clinical trial.Methods: ALRN-6924 is being evaluated alone and in combination with cytarabine, using a 3+3 dose escalation design. Using the previously determined RP2D for solid tumors (Meric-Bernstam et al., 2017), the initial cohorts receive 3.1 mg/kg of ALRN-6924 IV over 1 hour on Days 1, 8, and 15 of a 28-day cycle (QW mono-therapy). In separate cohorts, ALRN-6924 is combined with cytarabine IV over 1 hour (initially 100 mg/m2, later 200 mg/m2 ; QW combo-therapy). Later monotherapy cohorts receive more frequent ALRN-6924 administration on Days 1, 3, 5, 8, 10, and 12 of a 21-day cycle (TIW mono-therapy), with an initial dose level of 2.7 mg/kg.Adverse events (AEs) are assessed per CTCAE V4.03; responses are evaluated by the investigators according to IWG (Cheson 2006) and AML Response Criteria (Dohner 2010), for MDS and AML, respectively.Results: Preliminary results are being presented for 13 pts on QW mono- and 19 pts on QW combo-therapy. As of 13 July 2018, 32 QW pts have enrolled, with a median age of 75 years (38-90), 18 pts with de novo or secondary AML and 14 pts with MDS who failed hypomethylating agents. Thus far, pts have received 1-14 cycles.No DLTs have been observed and no MTD has been reached for QW mono-therapy (3.1-5.8 mg/kg) or QW combo-therapy (3.1-4.4 mg/kg ALRN-6924 and 100-200 mg/m2 cytarabine).For QW-treated pts (mono and combo), AEs related to treatment with ALRN-6924 have been reported in 62.5% of pts; most frequent are nausea (25.0%), thrombocytopenia (25.0%), vomiting (25.0%), fatigue (15.6%), and diarrhea (15.6%). Hyperbilirubinemia was reported in 18.8% of pts, representing a known transient effect of ALRN-6924 clearance that has not been associated with liver injury. Thrombocytopenia is the only ≥ grade 3 hematologic AE related to treatment with ALRN-6924 that was reported in ≥5% of pts, with all cases being grade 4 thrombocytopenia (7 pts, 21.9%). SAEs related to treatment with ALRN-6924 have been reported in 2 pts (grade 3 extremity pain and grade 4 angioedema while on concomitant lisinopril). The AE profiles for pts receiving ALRN-6924 QW mono- or QW combo-therapy were not significantly different.Including all QW cohorts (mono and combo), twenty-seven pts are efficacy-evaluable as of July 13, 2018. In preliminary efficacy results across all cohorts, marrow CR was reported in two out of four MDS pts treated with 4.4 mg/kg ALRN-6924 + 200 mg/m2 cytarabine, one of whom went on to transplant. An additional three MDS pts achieved SD. Among AML pts, 1 had a >50% reduction in bone marrow blasts while receiving 3.1 and then 4.4 mg/kg ALRN-6924 plus 100 mg/m2 cytarabine QW. This was durable until the pt succumbed to pneumonia that was unrelated to study drug in cycle 7.For QW-treated pts (mono and combo), main reasons for treatment discontinuation include treatment failure (31.2%), consent withdrawn (25.0%), death (9.4%), MDS transformed to AML (6.2%), and adverse event (6.2%).Accrual to QW combo-therapy with cytarabine in MDS and TIW mono-therapy in AML and MDS continues; updated data for QW mono-, QW-combo as well as TIW mono-therapy will be presented at the meeting.Conclusion: ALRN-6924 has shown clinical activity and an acceptable safety profile in AML and MDS. Continued development of ALRN-6924 for AML and MDS treatment is warranted. DisclosuresSallman:Celgene: Research Funding, Speakers Bureau. Borate:Agios: Consultancy; Novartis: Consultancy. Cull:Celgene: Speakers Bureau. Komrokji:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Steidl:Aileron: Consultancy, Research Funding. Corvez:Aileron Therapeutics Inc.: Employment, Equity Ownership. Payton:Aileron Therapeutics: Employment, Equity Ownership. Annis:Aileron Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. Pinchasik:Aileron Therapeutics Inc.: Employment, Equity Ownership. Aivado:Aileron Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties.

The Novel Small Molecule Inhibitor KS99 Targets AML and Inhibits Stemness Markers STAT3 and ALDH

Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be activated in leukemic stem cells (LSCs), especially in Pre-LSCs, and activation has been associated with poor prognosis in multiple cancers including AML. In AML, sorted CD34+/CD38- and aldehyde dehydrogenase-positive (ALDH+) cells have significantly phosphorylated STAT3 when compared to healthy controls. The Isatin analog, KS99 has shown selective anti-cancer activity against Multiple Myeloma (MM) which may, in part, be mediated by inhibition of BTK activation. Here we demonstrate that KS99 selectively targets AML CD34+/CD38- and ALDH+ cells and inhibits STAT3 phosphorylation while sparing normal hematopoietic stem and progenitor cells (HSPCs).Experimental Design: The pro-apoptotic activity of KS99 for primary human AML cells (n=21) and human AML cell lines (n=9) was evaluated using Annexin V and cell proliferation (MTS) assays. Colony forming assays were carried out to demonstrate the specific LSC-targeted effect of KS99. Flow cytometry was used to detect STAT3 phosphorylation and apoptosis in CD34+/CD38- LSCs and ALDH activity in a series of human AMLs. Next, to assess the preclinical efficacy, the human AML cell line U937 was injected intravenously (IV) into NSG mice. Mice with an established leukemic burden were randomized into treatment groups and treated through an intraperitoneal (IP) injection with either vehicle control or KS99 (2.5mg/kg) every other day x8. Two days after the final treatment, leukemic burden (hCD45+) was quantitated in the bone marrow.Results: AML cell lines exhibited dose-dependent sensitivity to KS99 in the nanomolar range (IC50: 150-400nM) as did most primary AML cells (Figure: A). Interestingly, poor prognosis AML subsets, AML with MDS related changes (MDS-RCs), were more sensitive than De Novo AML cases (p= 0.0077) with or without a NMP1 mutation (p= 0.012, p=0.045, respectively). Within De Novo AML, NMP1 wild-type cases were more sensitive than NMP1 mutant cases (p= 0.02) (Figure: B). Furthermore, KS99 treatment selectively reduced the clonogenicity of human AML patient cells vs. normal cord blood mononuclear cells (p<0.001) (Figure: C). KS99 induced apoptosis in the CD34+/CD38- gated cell population of primary AML, leading to reduced blast-like and stem-like cells, but not other cells. It also showed a reduction in ALDH+ cells in an Aldefluor assay. Likewise, KS99 treatment showed reduced pSTAT3 in viable CD34+/CD38- cells (Figure: D). Inhibition of pBTK, BCL-2, and pSTAT3 were seen in Western blot experiments. Animal studies showed 76 % reduction of hCD45+ U937 cells in bone marrow of KS99-treated mice when compared to bone marrow isolated from vehicle-treated animals (p<0.05) (Figure: E).Conclusion: KS99 selectively targets LSCs from poor prognosis AML patients while sparing normal HPSCs. This effect may be mediated via targeting of STAT3 signaling and ALDH, which are critical for proliferating LSCs, but not normal HPSCs. Further studies will aim to demonstrate mechanisms of activity and preclinical efficacy in patient-derived AML xenograft mouse models. [Display omitted] DisclosuresDovat:Elf Zone, Inc.: Membership on an entity's Board of Directors or advisory committees.

PO-395 Cortisol triggers immune escape of human acute myeloid leukaemia cells by inducing expression of G protein-coupled receptor latrophilin 1 which facilitates exocytosis of immune suppressor galectin-9

IntroductionAcute myeloid leukaemia (AML) is a blood and bone marrow cancer originating from self-renewing malignant immature myeloid cells. AML cells are capable of escaping host immune surveillance. One of the mechanisms underlying this phenomenon is the ability of AML cells to secrete galectin-9, a tandem protein, which induces variety of intracellular and cell-to-cell signalling events leading to inactivation of NK cells as well as killing of cytotoxic T cells. Exocytosis of galectin-9 is mediated by the G protein-coupled receptor latrophilin 1 (LPHN1). However, molecular mechanisms underlying its expression and functional control remain unknown, and thus became the main goal of our work.Material and methodsWe used THP-1 and primary human AML cells, primary leukocytes obtained from healthy donors. Western blot analysis, ELISA and quantitative Real-Time PCR served as main instrumental in experimental set-ups.Results and discussionsWe discovered that cortisol induces LPHN1 expression on transcriptional level thus also upregulating its translation in AML cells, but not in primary healthy human leukocytes. We found that secreted form of fibronectin and leucine-rich transmembrane protein 3 (FLRT3, an endogenous LPHN1 ligand) is present in blood plasma of healthy donors and AML patients in equally high amounts. Therefore, LPHN1 expressed by human AML cells is not lacking its ligand and thus triggers galectin-9 secretion on permanent basis in order to provide continuous protection against host immune surveillance.ConclusionOur results demonstrate a fundamentally novel survival mechanism employed by human AML cells. They use a crucial human hormone (cortisol) to express receptor (LPHN1), which uses blood plasma protein (FLRT3) to escape immune surveillance. Thus AML cells employ normal physiological systems of the human body to support their survival and attenuate anti-cancer activity of cytotoxic lymphoid cells.

Allosteric inhibitor remotely modulates the conformation of the orthestric pockets in mutant IDH2/R140Q

Neomorphic mutation R140Q in the metabolic enzyme isocitrate dehydrogenase 2 (IDH2) is found to be a driver mutation in cancers. Recent studies revealed that allosteric inhibitors could selectively inhibit IDH2/R140Q and induce differentiation of TF-1 erythroleukemia and primary human AML cells. However, the allosteric inhibition mechanism is not very clear. Here, we report the results from computational studies that AGI-6780 binds tightly with the divalent cation binding helices at the homodimer interface and prevents the transition of IDH2/R140Q homodimer to a closed conformation that is required for catalysis, resulting in the decrease of the binding free energy of NADPHs. If the allosteric inhibitor is removed, the original open catalytic center of IDH2/R140Q will gradually reorganize to a quasi-closed conformation and the enzymatic activity might recover. Unlike IDH2/R140Q, AGI-6780 locks one monomer of the wild-type IDH2 in an inactive open conformation and the other in a half-closed conformation, which can be used to explain the selectivity of AGI-6780. Our results suggest that conformational changes are the primary contributors to the inhibitory potency of the allosteric inhibitor. Our study will also facilitate the understanding of the inhibitory and selective mechanisms of AG-221 (a promising allosteric inhibitor that has been approved by FDA) for mutant IDH2.

Agonistic targeting of TLR1/TLR2 induces p38 MAPK-dependent apoptosis and NFκB-dependent differentiation of AML cells

AbstractAcute myeloid leukemia (AML) is associated with poor survival, and there is a strong need to identify disease vulnerabilities that might reveal new treatment opportunities. Here, we found that Toll-like receptor 1 (TLR1) and TLR2 are upregulated on primary AML CD34+CD38− cells relative to corresponding normal bone marrow cells. Activating the TLR1/TLR2 complex by the agonist Pam3CSK4 in MLL-AF9-driven human AML resulted in induction of apoptosis by p38 MAPK-dependent activation of Caspase 3 and myeloid differentiation in a NFκB-dependent manner. By using murine Trp53−/−MLL-AF9 AML cells, we demonstrate that p53 is dispensable for Pam3CSK4-induced apoptosis and differentiation. Moreover, murine AML1-ETO9a-driven AML cells also were forced into apoptosis and differentiation on TLR1/TLR2 activation, demonstrating that the antileukemic effects observed were not confined to MLL-rearranged AML. We further evaluated whether Pam3CSK4 would exhibit selective antileukemic effects. Ex vivo Pam3CSK4 treatment inhibited murine and human leukemia-initiating cells, whereas murine normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected. Consistent with these findings, primary human AML cells across several genetic subtypes of AML were more vulnerable for TLR1/TLR2 activation relative to normal human HSPCs. In the MLL-AF9 AML mouse model, treatment with Pam3CSK4 provided proof of concept for in vivo therapeutic efficacy. Our results demonstrate that TLR1 and TLR2 are upregulated on primitive AML cells and that agonistic targeting of TLR1/TLR2 forces AML cells into apoptosis by p38 MAPK-dependent activation of Caspase 3, and differentiation by activating NFκB, thus revealing a new putative strategy for therapeutically targeting AML cells.

Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells.

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

Pre-Clinical Activity of Novel Hypoxia-Activated FLT3 Inhibitors in FLT3-Mutated AML

Activating mutations in the Fms-like tyrosine kinase 3 (FLT3) are most frequently observed molecular abnormalities in AML, which lead to constitutive activation of the tyrosine kinase. While several mechanisms of resistance to FLT3 tyrosine kinase inhibitors (TKIs) have been identified, commonly failure is associated with lack of complete and sustained inhibition of FLT3 tyrosine kinase activity. FLT3 inhibition is particularly challenging in blasts within the hypoxic bone marrow (BM) microenvironment, which plays a crucial role in AML survival. Widespread hypoxia has been shown by us and others to be prevalent within the confines of the leukemic BM space, and to limit the efficacy of standard chemotherapy (Benito et al., PLoS One 6(8):e23108, 2011). With the goal of exploiting hypoxia as a physiological target and increasing selectivity to FLT3-mutant AML blasts, we have developed a series of hypoxia-activated prodrugs of FLT3 TKIs (HA-TKIs) based on the known clinical compounds sunitinib, AC220, MLN-518 and crenolanib. Bioreductive trigger conjugation was performed at the final step of the synthesis to provide the prodrugs as previously described (Lu et al., Tetrahedron, 69, p9130-9138, 2013). Under hypoxia, these compounds can undergo enzymatic one-electron reduction and then fragment selectively to release the cell permeable TKI, generating higher local concentrations of TKIs and limiting normal tissue exposures.Pulse radiolysis was used to determine the electron affinity (E(1)) and rate of fragmentation of each prodrug. The E(1) values ranged from -430 mV to -497 mV, while the rate of fragmentation of the prodrugs following one-electron reduction under hypoxia ranged from 28 s-1 to 400 s-1. SN37156 and SN37169 in particular demonstrated E(1) and kfrag parameters ideal for hypoxia-selective cellular metabolism (-449 mV, 85 s-1; -449 mV, 62 s-1, respectively).With the purpose of identifying the HA-TKIs with the most selective and dose-potent in vitro efficacy, we first performed an Oxic/Hypoxic screening of the hypoxia-dependent activity of HA-TKIs against FLT3-mutant AML cell lines. From the 10 compounds originally developed, SN37156 and SN37169 showed hypoxia-dependent apoptosis and growth arrest. SN37156 demonstrated the best activity against Molm13, Molm14 and MV4-11 cells under hypoxia with hypoxic cytotoxicity ratios (oxic/hypoxic IC50 ratios) of 4.9, 11 and 17.6, respectively.In our secondary screening we compared the activity of SN37156, SN37169 and their parental inhibitors on target modulation. Both HA-TKIs selectively inhibited FLT3 and ERK auto phosphorylation in MV4-11 cell line under hypoxic but not oxic conditions, with similar potency to the parental inhibitor under normal oxygen culture conditions.We next investigated the effects of both HA-TKIs on inhibition of FLT3 downstream targets, including AKT, ERK, pS6 and STAT5 using time-of-flight mass cytometry (CyTOF). The data demonstrated distinct patterns of signaling inhibition, with a dose-dependent p-ERK and p-S6 inhibition in MV4-11 and Molm-14 cells upon treatment with both HA-TKIs. In turn, both compounds at 100nM inhibited p-AKT and p-STAT5.Single dose tolerability studies were conducted in tumor-free C57BL6 mice. Prodrugs were administered by IP injection in 20% β-hydroxypropylcyclodextrin solution starting at 17.8 µmol/kg and escalating in 1.33-fold increments until lethality or severe mortality (mean body weight loss of >10%) was observed. The maximal tolerated dose for both, SN37156 and SN37169 was established at 56.1 µmol/kg. To test efficacy of HA-TKIs in vivo, we injected C57BL6 mice with genetically engineered Baf3-ITD/luc/GFP cells. Mice were randomized to treatment groups on day 8 when engraftment was documented by bioluminescent imaging (BLI), and treated with either vehicle or 56.1 µmol/kg of SN37156 or SN37169 I.P. every 3 days. Multiple doses (up to 10 so far; study is ongoing) were well tolerated. BLI demonstrated reduced leukemia burden in both treatment groups compared to control.In summary, our data indicate that HA-TKIs SN37156 and SN37169 inhibit mutant FLT3 and its selected downstream signaling targets under conditions resembling marrow hypoxia, and induce hypoxia-dependent growth arrest and apoptosis in FLT3-mutant AML cells. Planned studies in FLT3-mutant AML PDX will evaluate in vivo efficacy to eliminate primary human AML cells from hypoxic BM microenvironment. DisclosuresDaver:Kiromic: Research Funding; Ariad: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; Otsuka: Consultancy, Honoraria; Sunesis: Consultancy, Research Funding. Konopleva:Cellectis: Research Funding; Calithera: Research Funding.

RNAi profiling of primary human AML cells identifies ROCK1 as a therapeutic target and nominates fasudil as an antileukemic drug

AbstractAcute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral loss-of-function RNA interference (RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was toxic to this cell fraction in feeder coculture and xenotransplant experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient–derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling.

Targeted Suppression of Interleukin-1 Signaling Attenuates Leukemic Cellular Growth and Disease Progression in Primary Human Cells and Murine Models of Acute Myeloid leukemia­­

Background: Despite the great strides that have been made in the treatment of AML, resistance to treatment is common and signaling pathways influencing leukemic cellular proliferation have not been comprehensively defined. Cytokines and growth factors play important roles in cell survival, proliferation, differentiation, senesence, and immune response in both normal and cancer cells. Recent studies have demonstrated that certain inflammatory cytokines known to suppress normal hematopoiesis can have the opposite effect (i.e. enhancement of proliferation) on cell growth of AML cells. To test the importance of this mechanism, we systematically assessed the functional relevance of 98 cytokines and their receptors in primary human AML cells and identified interleukin-1 (IL-1) and its receptor as critical determinants in aberrant regulation of AML cell growth.Methods: To identify functionally important cytokine signaling pathways in AML pathogenesis we first quantified the proliferation of 50 primary AML patient samples in the presence of graded concentrations of 98 cytokines. We then employed a functional siRNA screen targeting 188 cytokine and growth factor receptors found to be highly expressed in 140 primary leukemia samples by gene expression analysis. Cytokine pathways of interest were functionally tested for their role as potential therapeutic targets utilizing primary patient samples and in vivo murine models.Results: We found that 40% of primary AML samples exhibited a 3- to 20-fold increase in cellular growth and a 2-fold decrease in apoptosis in the presence of IL-1α/β. Paradoxically, IL-1 suppressed the growth of normal CD34+ cells. Silencing of the IL-1 receptor, IL1R1, reduced the viability of these AML primary samples by 60-80%. Notably, most of the IL-1-sensitive AML samples exhibited monocytic and myelomonocytic features. To demonstrate the importance of IL-1 signaling in the survival of AML cells, we utilized IL1R1-/- mice and oncogene-induced leukemic cells in vitro and in vivo. We observed that IL1R1-/- mice and wild-type mice showed a normal distribution of stem and progenitor populations in their marrow by FACS analysis. Further, the absence of IL1R1 in murine bone marrow leads to a significant ablation of clonogenic potential (80% reduction) of oncogene-induced leukemic cells (AML1-ETO9a, NRASG12D and MLL-ENL) when compared to oncogene-induced leukemic cells from wild-type mice in a ligand-dependent manner. No difference in colony growth was observed with empty vector-transduced marrow cells. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer for a median of 39 days (range: 28-118) compared to 30 days (range: 27-61) for recipients of wild-type marrow (p=0.012). Mice transplanted with IL1R1-/- marrow or wild-type marrow showed comparable white blood cell and platelet counts. However, histopathological analysis showed a significant reduction in myeloid infiltrates in liver and lungs as well as reduced marrow cellularity and reticulin fibrosis in IL1R1-/- leukemic mice as compared to wild-type leukemic mice. To exclude the possibility that this difference was related to differences in homing and engraftment of wild-type and IL1R1-/- bone marrow cells, we compared short-term homing and long-term engraftment of empty vector-transduced wild-type and IL1R1-/- marrow cells and found no significant differences. These results demonstrate an in vivo role of IL1-receptor in AML cell proliferation and progression. Mechanistically, exogenous IL-1 promotes the growth and survival of primary CD34+ AML cells and AML cell lines by increasing p38 MAPK phosphorylation. Conversely, knocking down IL1R1 or treating AML cells with p38 kinase inhibitors such as doramapimod (n=10; median IC50: 70 nM) reduced the growth of AML cells by decreasing phosphorylation of p38 kinase. No toxic effect of doramapimod was observed on normal cell growth, suggesting that targeting p38 kinase might be therapeutically beneficial for a subset of AML patients dependent on IL-1 signaling.Conclusion: These results demonstrate a novel in vitro and in vivo role for IL-1 and its receptor in promoting leukemic cellular growth and progression in a large subset of AML patients and warrant further investigation of this pathway as a therapeutic opportunity. DisclosuresNo relevant conflicts of interest to declare.

The Broad Activity of the CD33/CD3 Bispecific BiTE® Antibody AMG 330 in Primary Human AML Is Impacted By Disease Stage and Cytogenetic/Molecular Risk

Background: Recent studies have demonstrated that AMG 330, a novel CD33/CD3-directed bispecific T-cell engaging (BiTE¨) antibody, is highly active against CD33+ AML cell lines and can potently lyse leukemic blasts from AML patients. Here, we have investigated the preclinical determinants for AMG 330 activity in primary newly diagnosed and relapsed/refractory human AML samples to prospectively understand factors that might contribute to clinical response or resistance.Patients and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML who consented to their use for research purposes. CD33 expression on myeloblasts (identified by appropriate CD45 and side-scatter properties) and the percentage of endogenous CD3+ T-cells were quantified by flow cytometry. To determine drug-induced cytotoxicity, AML cells were incubated in culture medium containing various concentrations of AMG 330; in some experiments, exogenous T-cells (isolated via magnetic cell sorting from a healthy adult volunteer who underwent leukapheresis) were labeled with CellVue Burgundy and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; AML cells were identified by forward/side scatter properties and negativity for CellVue Burgundy dye. Specific drug-induced cytotoxicity was calculated as: 100 x (1 – live target cellstreated/live target cellscontrol), and presented as mean±SEM.Results: Forty-one of the 49 studied specimens were included in our analyses as they had >40% myeloblasts upon thaw and were >50% and >30% viable at baseline and after 48 hours, respectively. Median age of the patients was 65.3 (range: 23.9-80.0) years. The median percentage of myeloid blasts and CD3+ T-cells in the studied specimens was 87.1% (range: 55.1-97.0%) and 2.0% (range: 0-27.3%), respectively, and median sample viability after 48 hours in culture was 76.7% (range: 31.1-93.5%). These characteristics were similar between the newly diagnosed (n=21) and relapsed/refractory (n=20) AML specimens. In the absence of healthy donor T-cells, AMG 330 resulted in modest cytotoxicity (at 500 pg/mL: 11.8±2.5%) that was correlated with the amount of autologous T-cells (at 500 pg/mL; r=0.566; p=0.0001; Spearman’s rank correlation test). On the other hand, AMG 330 exerted marked cytotoxic activity in several specimens with very low CD33 expression, and there was no correlation between AMG 330 cytotoxicity and CD33 expression (at 500 pg/mL; r=-0.048; p=0.77). In the presence of healthy donor T-cells, AMG 330 cytotoxicity was strictly dependent on the drug dose (e.g. p<0.0001 at E:T=1:3) and E:T cell ratio (e.g. p<0.0001 at 500 pg/mL; Fig. 1). High cytotoxic activity of AMG 330 was observed in specimens with very low CD33 expression, and a correlation between AMG 330 cytotoxicity and CD33 expression was only observed at high E:T cell ratio (3:1) and higher AMG 330 doses (at 500 pg/mL: r=0.465, p=0.0022). In analyses of patient subsets, AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML (n=21) than those with relapsed/refractory disease (n=20) despite similar levels of CD33 on leukemic blasts (median of 837 [30-5,356] vs. 1,018 [7-2,567] arbitrary fluorescence units; Fig. 2A and B). Furthermore, AMG 330-induced cytotoxicity was higher in specimens from patients with cytogenetically/molecularly favorable-risk disease (n=5) as compared to those with intermediate- (n=26) or adverse risk disease (n=10; Fig. 2C and D).Conclusion: AMG 330 causes potent cytolysis of primary human AML cells in vitro in a dose- and E:T cell ratio-dependent manner across the entire spectrum of cytogenetic/molecular risk and disease stage, even in specimens with very low expression of CD33. Lower activity of AMG 330 was observed in relapsed/refractory AML specimens (relative to newly diagnosed AML specimens) and intermediate- or adverse-risk disease specimens (relative to favorable-risk specimens) suggesting the potential presence of yet undefined, CD33-independent, relative resistance mechanisms in defined patient subsets.Figure 1 [Display omitted] Figure 2 [Display omitted] DisclosuresOff Label Use: Use of AMG 330 in AML (AMG 330 is investigational drug). Newhall:Amgen, Inc: Employment. Sinclair:Amgen, Inc: Employment. Frankel:Amgen, Inc: Employment. Kischel:Amgen, Inc: Employment. Chen:Amgen, Inc: Employment. Walter:Amgen, Inc: Research Funding; Seattle Genetics, Inc.: Research Funding; Amphivena Therapeutics, Inc.: Consultancy; Covagen AG: Consultancy.

Gain-of-Function Mutant p53 Enhances Hematopoietic Stem Cell Self-Renewal

The tumor suppressor p53 is a critical regulator of hematopoietic stem cell (HSC) behavior and we demonstrated that p53 maintains HSC quiescence and regulates HSC response to irradiation (Liu et al., Cell Stem Cell, 2009).While TP53 mutations are less common in acute myeloid leukemia (5 to 8%) than in solid tumors (50%), they are associated with poor prognosis and abnormal cytogenetics, especially abnormalities in chromosomes 5 and 7. These mutations may abolish some but not necessarily all of the functions of p53 in regulating stem cell behavior. Therefore, this aspect of p53 function needs further investigation.To define the role of mutant p53 in the pathogenesis of AML, we introduced 9 hot-spot p53 mutants identified in AML patients, including p53R248W, p53R273H and p53Y220C, into wild type hematopoietic cells using retrovirus-mediated transduction and investigated the role of these p53 mutants in regulating HSC self-renewal. We found that hematopoietic cells expressing p53R248W, p53Y220C or p53R273H show enhanced repopulating potential 16 weeks following transplantation. As codon 248 of the p53 protein is most frequently mutated in AML, we decided to investigate the role of p53R248W mutant in HSCs by using the humanized knock-in mice of p53R248W. In p53 knockout mice, there is a dramatic increase of HSCs (CD48-CD150+Lin-Sca1+c-Kit+ cells); however,we found thatboth wild type andp53R248W mice have similar number of HSCs. While wild type p53 maintains HSC quiescence, expression of p53R248W in HSCs (CD48-CD150+LSKs) does not affect their quiescent state. Asp53R248W does not appear to affect HSC frequency and quiescence, it is not a loss-of-function mutant. We also used bone marrow cells isolated from both wild type and p53R248W mice to perform the serial replating assays and found that expressing p53R248W from the endogenous Trp53 promoter enhances the replating potential of hematopoietic cells. Moreover, we performed serial bone marrow repopulation (BMT) assays and found that the repopulating ability of p53R248W cells was significantly higher than that of the wild type cells in both primary and secondary BMT assays, demonstrating that the p53R248W mutant enhances HSC self-renewal in vivo. Furthermore, we observed that HSCs expressing p53R248W are resistant to genotoxic stress induced by irradiation and the p53R248W mice show extended survival following sub-lethal dose of total body irradiation. Ample data indicate that mutant p53 proteins not only lose their tumor suppressive functions, but also gain new abilities that promote tumorigenesis. To understand how mutant p53 enhances HSC self-renewal, we performed gene expression profiling assays by using HSCs isolated from wild type and p53R248W mice. We also utilized Ingenuity Pathway analysis software to group putative mutant p53 target genes into different pathways. While we did not observe change in the expression of p53 target genes in p53R248W HSCs, several pathways that are important for leukemogenesis, including epigenetic and DNA damage repair pathways, are altered in HSCs expressing p53R248W, demonstrating that p53R248W is a gain-of-function mutant. Given that TP53 mutations are correlated with poor prognosis, pharmacological inhibition of mutant p53 may be a promising therapeutic strategy for AML patients with TP53 mutations. Small molecule PRIMA-1 has been shown to restore wild-type conformation to some mutant p53 proteins and induce apoptosis in human tumor cells. We found that hematopoietic cells expressing mutant p53 are sensitive to PRIMA-1 treatment and undergo p53-dependent apoptosis. Furthermore, we observed that PRIMA-1 inhibits the growth of primary human AML cells with TP53 mutation in a dosage-dependent manner.Taken together, we demonstrated that gain-of-function mutant p53 enhances hematopoietic stem cell self-renewal through regulating epigenetic and DNA damage repair pathways. Our data also suggest that pharmacological inhibition of mutant p53 may sensitize the drug-resistant leukemia stem cells (LSCs) to chemotherapy and improves leukemia treatment. DisclosuresNo relevant conflicts of interest to declare.

Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells

The histone demethylase LSD1 (KDM1A) demethylates mono- and di-methylated (Me2) lysine (K) 4 on histone H3. High LSD1 expression blocks differentiation and confers a poor prognosis in AML. Here, treatment with the novel LSD1 antagonist SP2509 attenuated the binding of LSD1 with the co-repressor CoREST, increased the permissive H3K4Me3 mark on the target gene promoters, and increased the levels of p21, p27 and C/EBPα in cultured AML cells. Additionally, SP2509 treatment or LSD1 shRNA inhibited the colony growth of AML cells. SP2509 also induced morphologic features of differentiation in the cultured and primary AML blasts. SP2509 induced more apoptosis of AML cells expressing mutant NPM1 than MLL fusion oncoproteins. Treatment with SP2509 alone significantly improved the survival of immune-depleted mice following tail-vein infusion and engraftment of cultured or primary human AML cells. Co-treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) and SP2509 was synergistically lethal against cultured and primary AML blasts. Compared to each agent alone, co-treatment with SP2509 and PS significantly improved the survival of the mice engrafted with the human AML cells, without exhibiting any toxicity. Collectively, these findings show that the combination of LSD1 antagonist and pan-HDI is a promising therapy warranting further testing against AML.

A Genome Wide shRNA Screen In Primary Human AML Cells Identifies ROCK1 As a Novel Therapeutic Target

In spite of recent advances, the prognosis especially of elderly AML patients remains unsatisfactory with survival rates of less than 10 % at 10 years. Genome-wide RNA-interference screens systematically interrogating the specific vulnerabilities of leukemic cells could be a valuable tool to identify novel therapeutic targets in this patient population. So far, such screens have only been done in immortalized cell lines and / or at sub-genome scale, which limits their transferability to individual patients.Therefore, we set out to establish an unbiased genome-wide pooled shRNA screen in primary human AML cells to prove the feasibility and test the possible clinical implications of such an approach.Lentiviral transduction of primary leukemic blasts from a 67-year old patient with AML FAB M1 with a pooled shRNA library (Mission TRC shRNA library SP1, Sigma) according to a specifically optimized protocol resulted in a transduction rate of 25 %, thus rendering multiple integrants unlikely. An aliquot of the cells was separated for DNA extraction directly after removal of viral supernatant (day 0) and after 9 days of suspension culture (day 9). ShRNA barcodes integrated into the genome of the host cells were read out using PCR-coupled next-generation sequencing (HiSeq 2000, Illumina). Of 7709 shRNA contained in the library, 6626 were recovered with at least 10 reads in the day 0 sample. After 9 days of culture, 25 shRNA targeting a total of 12 genes were identified as potentially lethal to the patient's AML-cells (Table 1). All of these shRNA were subjected to single-shRNA transduction experiments using leukemic cells from the same donor. In fact, 18 of 25 shRNA were validated with respect to viability. Knockdown specificity was documented for all validated shRNA by qPCR. For further analyses we focused only on those 7 genes in which more than 50% of the shRNA identified in the pooled screen could be validated (Table 1). These genes were assessed for druggability using publicly available databases. For exploration of the potential therapeutic implications of our screen we chose ROCK1 as a potential target, because Fasudil, a specific ROCK1 inhibitor, has already been licensed for the treatment of pulmonary hypertension in humans.TableTable1No. shRNAs >100 reads day 0No. scoring shRNA in pooled screenNo. valdiated shRNA in single shRNA experimentsOverall gene validation statusBNIPL322ValidatedC7orf16322ValidatedCCRL1321Not validatedDGAT2321Not validatedDUSP14320Not validatedMAP3K6422ValidatedROCK1532ValidatedRPS13322ValidatedSF3A1321Not validatedSNX27422ValidatedSTK3422ValidatedWDHD1220Not validatedKnockdown of ROCK1 in primary leukemic blasts led to rapid cell-cycle arrest and cell-death. Treatment with Fasudil proved to be equally effective in killing leukemic cells. Compared to primary leukemic cells from the original as well as from other AML patients, Fasudil seemed to be less toxic to hematopoietic cells derived from healthy volunteer donors. RNA-sequencing revealed that in comparison to the healthy controls none of the studied AML patients demonstrated a significant overexpression of ROCK1. Moreover there was no indication for a functional ROCK1 mutation in the analyzed AML samples. Feeder based long-term culture initiating cell (LTC-IC) assays further suggested that Fasudil had a significant negative effect on the self-renewal capacity of primary human leukemic stem/progenitor cells (Figure 1). Studies in xenograft-models to assess the stem cell toxicity of ROCK1 inhibition in more detail are currently ongoing. [Display omitted] Taken together our results show that pooled shRNA screens in primary patient-derived leukemic cells are feasible and able to pinpoint novel therapeutic targets, which might be missed in mutation- or overexpression-based approaches. Further optimization of transduction and screening protocols might enable such screens to assist physicians in the selection of optimal therapeutic strategies especially in poor risk AML. Disclosures:No relevant conflicts of interest to declare.