ALBERT

Description: Enhanced blast clearance/remission followed for relapsed/refractory AML patients, in whose blasts were annotated by mutant Flt3 in company with additional TET2 mutation, when a combination of Sorafenib/Vorinostat was supplemented by Bortezomib (Sayar, et al. Oncotarget, 2018). Pharmacodynamic analysis of day0/4 marrow blasts demonstrated early inhibition of HOXA9/10, and/or MEIS1, which occurred upon re-regulation of Wnt pathway participants whose activity had been affected by mTET2. We hypothesized Wnt effectors which drive HOXA overexpression enlist β-Catenin-dependent transactivation of HOXA (Bei, Eklund et al. JBC, 2012), and the role of Bortezomib extends beyond previously recognized AML-specific targets: Flt3ITD, p52NFκB. Therefore a series of primary AML blasts chosen across a spectrum of cytogenetic and molecular categories were analyzed herein to examine the hypothesis that proteasome inhibitors (PI) provide novel targeting action in combination with a new Flt3/Syk inhibitor (FSI) currently in clinical trial, TAK-659 (K Pratz, Blood(abstract) 2018) or, alternatively, when using a previously-reported compound with similar FSI selectivity, R406 (Puissant, Stegmaier, et al. Cancer Cell, 2014). Either of these agents alone or in combination with Bortezomib or Ixazomib were tested to learn respective mechanism of action, and to identify sensitive molecular phenotypes. In order to study β-Catenin as target, we incubated AML blasts in culture with the following treatments: control, FSI, PI, or PI+FSI, while using informative dosing in tandem cultures for proliferation, apoptosis, gene expression and proteomic endpoints. Because ubiquitination directs substrate proteolysis as well as subcellular localization for transcriptional regulators, we isolated within each test group both cytoplasmic and nuclear protein. Also, because activity of β-Catenin involves phosphorylation-initiated and ubiquitin-directed trafficking, we probed immunoblots for both nonphosphorylated [active] (S33,S37,T41)- and phosphorylated β-Catenin species. For Flt3/Syk inhibition by TAK-659, we used concentrations at, and below, a clinically-achievable optimal concentration of 250 nM (opcit). In all cases of Flt3mutant AML's studied, we observed synergy between Flt3/Syk inhibitor with PI, including at clinically suboptimal concentrations of individual agents. This cooperative activity involved loss of active, nonphosphorylated β-Catenin (80kD) from the nucleus, and the cytoplasmic accumulation of large ubiquitin-laden species of phospho-β-Catenin, ranging from 100-175kD, under conditions where apoptotic cell death occurred. A cytoplasmically-localized gatekeeper for stopping canonical Wnt pathway activation, Axin1, was not limiting for the loss of nuclear β-Catenin by the combination: FSI plus PI, but was modestly upregulated by PI alone. However, nuclear-localized FOXM1, a known nuclear chaperone for β-Catenin, was frequently significantly reduced by PI or the combination of FSI/PI. Both Flt3ITD (cytoplasmic) and p52NFκB (nuclear) were lost in additive fashion by combination treatments. At the transcriptional level (real-time RT-PCR), c-jun (a transactivator for FOXM1 and CTNNB1), Syk, as well as FOXM1, HOXA and MEIS1 were additively repressed by combination FSI plus PI in select AML blasts. Certain AML molecular phenotypes demonstrated heightened sensitivity to the combination of TAK-659 plus PIs. Within blasts bearing hierarchically-related mutant effectors: IDH2, TET2, or WT1-all recognized to dampen Wnt-inhibitory pathways, we observed that the combination of TAK-659 with PI was cooperatively active in both Flt3mutant and Flt3wild-type AMLs, while accompanied by an inhibitory effect on β-Catenin. In summary, proteasome inhibition with Ixazomib or Bortezomib cooperated with Flt3/Syk inhibition to cause apoptotic cell death in both Flt3mutant and wild-type AML's with Wnt pathway DNA/chromatin modifying enzyme mutations, a process which appeared to depend upon loss of nuclear-active β-Catenin and FOXM1, and accompanied by accumulation of large, ubiquitin-laden cytoplasmic β-Catenin species. Thus, proteasome inhibition in combination with inhibition of Flt3/Syk holds a central role in targeted therapy of certain poor-risk de novo AMLs, by interdiction of a leukemic stem cell (LSC) signature and causing apoptosis of AML blasts. Disclosures Roodman: Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3272 Epigenetic repression of tumor suppressor genes, including DAPK1, p16INK4a, and RUNX3, cooperates with survival signaling to confer adverse prognosis in high-risk AML, including normal karyotype (NK) Flt3ITD. Cyclic administration of the multikinase inhibitor sorafenib, whose targets include Flt3 and raf/MAPK, along with vorinostat, a pan-HDAC inhibitor, was undertaken in a group of patients with relapsed/refractory AML or older than 70. Pharmacodynamic monitoring was performed for molecular determinants of response, operating from a functional platform for communication by tyrosine kinase survival pathways and downstream epigenetic targets: Flt3-to-p52NFkB/histone deacetylases are implicated in DAPK1 repression; Flt3ITD-to-p52NFkB/stat5-to-pim1 suppresses endoplasmic reticulum (ER) stress via DAPK1 repression/4-EBP1 inhibition; Flt3-wild-type or/ITD-induced Id1 is linked to p16INK4a repression. Using a cyclic 21-day schedule consisting of 14 days of treatment followed by 7-days rest, sorafenib was given orally at 400 mg bid, and oral vorinostat administered with escalating doses in a 3×3 cohort design: dose levels of 200 mg, 300 mg, and 400 mg daily in two divided doses. Response was assessed by standard clinical parameters, including bone marrow examination at day 15. No DLTs were observed in the first two cohorts. One/six patient in the third cohort developed grade 4 diarrhea prompted by neutropenic sepsis. Three additional patients were treated at this dose, with no DLTs. The optimal dose was established as sorafenib 400 mg bid and vorinostat 200 mg bid for 14 days followed by 7 days rest. Among a total of 15 treated patients, 13 were evaluable for response. Six/thirteen (46%) patients demonstrated PR with the first cycle of treatment, and 1/13 (8%) achieved a CR with one cycle. Two/six (33%) very good partial responders and one unevaluable patient had NK Flt3ITD; all demonstrated complete clearance of peripheral blood blasts within 1 week, starting as high as 52–84,000/uL; one had reduction of marrow blasts to 10% (from initial 90%) after 2 cycles. Surprisingly, molecular phenotypes of the 3 Flt3ITD+ patients differed: pre-treatment blasts in one patient strongly expressed nuclear phospho-c-jun, p52NFkB, phospho-stat5, and Id1, with transcript levels of meis1, hoxA9, and pim2 at greater than 3-log10 by RQ-PCR. At day 3, 2–3 log reduction of hoxA9, meis1, and pim's1/2 were recorded prior to morphologic change. No epigenetic upregulation of p16INK4a or DAPK1 was observed, but an upregulation of (inactive) surface Flt3 and IRE1a, and cleavage of pro-caspase 4 indicated an evolving ER stress apoptosis. Another Flt3ITD+ patient's pre-treatment blasts had evidence of an epigenetic signature with repression of RUNX3, DAPK1, and p16INK4a transcripts, while nuclear p52NFkB/relB and stat5 were abundant by immunoblot, but transcript levels for pim1/2 were not elevated. At day 4 of therapy, again there was evidence for evolution of an ER stress apoptosis pathway, but in association with upregulation of DAPK1 and RUNX3 transcripts by 5 and 7 fold, respectively. However, hoxA9 and meis1 transcripts were not reduced and pim1/2 levels rose. With protocol-defined failure to improve at day 42, pim1/2 levels continued to rise. This patient only had mutant Flt3 alleles and most Flt3 protein was nonglycosylated/ER-internalized, which supports an evasion mechanism dependent on ER-localized Flt3ITD stat5 signaling. The patient who achieved CR, carried complex cytogenetics. Blasts had high pre-treatment Id1 expression with repression of tumor suppressors p16INK4a and RUNX3, and 2-log upregulation of p16INK4a/RUNX3 occurred at day 4. By contrast, in another patient of complex cytogenetics without CR, p16INK4a upregulation did not occur. Patients with response demonstrated depletion of p52NFkB at day 3/4. Lack of p52NFkB abrogation signaled treatment failure. Of particular interest, in vitro exposure of pre-treatment blasts of almost all patients to combination of sorafenib and SBHA (a vorinostat analogue) largely predicted clinical efficacy. In summary, the combination was safe and had clinical activity, and pharmacodynamic monitoring revealed unexpected heterogeneity within classically-defined molecular phenotypes. In addition, findings support the possibility of significantly improving responses by addition of a direct inducer of ER stress, bortezomib, to the combination. Disclosures: Sayar: Onyx/Bayer: Research Funding. Off Label Use: A trial of sorafenib and vorinostat in AML based on previously demonstated independent single-agent activity in the disease.

Description: Abstract 1660 GSEA (gene-set enrichment array) analysis for expression levels in acute myeloid leukemia (AML) blasts of hoxA9/meis1, has afforded additional prognostic capacity to cytogenetically-defined subtypes. Signaling pathway (KEGG, GO, Ingenuity) analysis of Affymetrix gene expression data has also been used to predict prognosis of AML patients, identifying an adverse high MAPK gene signature. Genome-wide epigenetic (DNA methylation) profiling has added to AML prognostication. We analyzed patient prognosis and in vitro sensitivity for blasts by pathway-targeting agents, in relation to expression clusters defined by the results of GSEA profiling. Blast cell sensitivity was assayed using the agents: (Tyrosine kinase inhibitors for: Flt3: Sorafenib (Onyx/Bayer) and Syk/Flt3: R406 (AstraZeneca); Bortezomib (Millenium), a proteasome- and NFkB- inhibitor, inducer of endoplasmic reticulum (ER) stress; or the pan-histone deacetylase (HDAC) inhibitor, SBHA, Vorinostat analogue). A group of 70 AML cases was studied. GSEA's (Taqman/RQ-PCR, ABI) were performed on two different 31-gene platforms interrogating the interaction of tyrosine kinase survival pathways (eg. Flt3) with downstream epigenetic targets: tumor suppressor genes DAPK1, p16INK4a/CDKN2A, and RUNX3. Expression levels of these are responsive to input from c-jun/AP-1, non-canonical NFkB isoforms/HDAC's, polycomb genes, and ets/ERG, which were monitored. Analysis of overall patient survival (OS) by Kaplan-Meier plots revealed conformity with established outcomes for conventional cytogenetic categories. Also, poor-risk categories defined by normal karyotype Flt3ITD mutation, as well as high hoxA9 and meis1 expressions were recognized. Classification categories involving NKFlt3ITD/high hoxA9/meis1 or complex cytogenetics/high MAPK were most strictly separated by Kaplan-Meier curves, and by representation on the GSEA heatmaps, vs. CBF/PML-RAR translocations. Specific association, between repression of the tumor suppressor DAPK1 (normalized to c-jun, a transcriptional activator) with high expressions of hoxA9 and DAPK1-repressive, non-canonical NFkB/relB, was apparent in most NKFlt3ITD and tMLL cases. Also, a known functional interaction implicating high Id1 expression with its repression of p16INK4a(CDKN2A) was linked by their expression levels on GSEA. The DAPK1/CDKN2A clusters demonstrated significant overlap, often with additional RUNX3 repression. The DAPK1-repressed(R) clusters demonstrated independent prognostic importance. There was median OS for DAPK1-R cluster #1(lower MAPK signature) of 17 months vs. for DAPK1-R cluster #2 of 6 months. This latter cluster was described by higher MAPK signature evidenced by heightened transcript levels of BRCA1 [Bullinger et al. Blood, 2007], FoxM1, bcl-2, IL-1b. The dominant cytogenetic/molecularly-defined phenotypes represented in these DAPK1-R clusters were NKFlt3ITD+ve and tMLL. A very-high MAPK/complex cluster, evidenced median OS 4.5 months vs. good prognosis (low MAPK/DAPK1, and low hoxA9/meis1) group: median OS〉 3 years. Interestingly, in vitro activity for the dual inhibitor R406 on blasts was greatest in the category with strongest repression of both p16INK4a/CDKN2A and DAPK1, and with lowest expression of syk transcripts (10-fold compared with high patient values), with a median IC50 of between 10 nM (tMLL)-100nM (Flt3ITD). By contrast, Sorafenib demonstrated its greatest activity in a high MAPK phenotype, including but not restricted to Flt3ITD+ve status. Bortezomib demonstrated median IC50 40 nM. However, high expression levels of FoxM1, putative Bortezomib target, were negatively-associated with its activity. SBHA, the HDAC inhibitor, demonstrated lower activity on a molar basis, but had important synergy with tyrosine kinase inhibitors, a property that was optimal in the context of Id1 hyperexpression. Bortezomib was strongly synergistic with Flt3/syk inhibitors, particularly on FltITD+ve blasts. Indeed, both Bortezomib and the HDAC inhibitor demonstrated by correlation analysis (coefficients=63.3 and 13.4, respectively) optimal activities with Id1 hyperexpression. In conclusion, a pathway-focused genetic and epigenetic prognostic classification that also reports targeting agent sensitivity was established and further validated in a phase I (Sorafenib/Vorinostat) trial in AML (H Sayar, these abstracts). Disclosures: Sayar: Onyx/Bayer: Research Funding.

Description: Abstract 2387 Poster Board II-364 Treatment failure in AML has been linked with the consequences of Flt3 signaling, although effectors of such resistance are uncertain. However, poor prognostic Flt3-ITD-containing AML's and some other intermediate prognostic groups have been found to have blunted spontaneous unfolded protein response/ER stress apoptotic pathway (Schardt, et al. Clin Canc Res. 15: 3834, 2009) that correlates with adverse outcome of therapy. We investigated the origins of suppressed ER-stress apoptosis in poor-risk AML by assaying a rate-limiting enzyme effector, DAPK1. Real-time RT-PCR analysis of DAPK1 expression, as well as its putative transcriptional activator(s), including c-jun, and its transcriptional repressor(s), including relB, and a gene-set enrichment array (GSEA) of NFkB- and c-jun-responsive (cytokine) genes, was undertaken in 30 AML samples. This included a control cohort of good-prognosis CBF+ve patients. A group of poor prognosis patients, especially those with normal karyotype and Flt3 (ITD) mutation, and those with MLL translocations, had on-average 10-fold (range 2-100-fold) reduction of DAPK1 transcripts, when normalized to expression of c-jun, a transcriptional activator of DAPK1, or to Meis1, another c-jun transcriptional target, which is a validated AML adverse prognostic array gene. Indeed, NK Flt3-ITD or t-MLL AML blasts with DAPK1 repression were characterized by exclusive nuclear presence of p52NFkB/relB, known to attract epigenetic and repressive components, including histone deacetylase enzymes (HDAC's) to the tandem CRE/NFkB sites (“integrated circuit”) at 5' DAPK1 -177bp and -134bp, respectively (Puto and Reed, Genes&Dev 22:998, 2008; Huang et al. Mol Cell 35: 48, 2009; Gade et al. Mol Cell Biol. 28: 2528, 2008). In the t-MLL/Flt3-ITD AML cell line MV-4-11, knockdown of NIK, IKK1, or p52NFkB2, while sparing Flt3-to-JNK/c-jun signaling, sharply upregulated (3-5-fold) DAPK1 expression. Flt3 inhibition in primary AML cells by using R406 (Rigel Pharmaceuticals, also a lead compound in CLL/NHL) a selective tyrosine kinase inhibitor that affects Flt3 auto-phosphorylation (and syk with similar cellular IC50's) also de-repressed DAPK1 expression in AML blasts. This outcome was associated with an inhibition of NIK and p52NFkB (both were also inhibited by Flt3 knockdown in MV-4-11) and a rise in apoptosis in R406-treated primary AML cells occurred. The combination of R406 with an HDAC inhibitor, suberoyl bis-hydroxamic acid (SBHA) showed synergistic inhibition of proliferation (tritiated thymidine incorporation). ER stress pathway activation evidenced by IREalpha and p-eIF2alpha upregulation was enhanced in primary AML blasts. Bortezomib (a proteasome-targeted inhibitor of NFkB/ER stress potentiator) also strongly inhibited p52NFkB activity and induced apoptosis. These studies reveal that transcriptional/epigenetic suppression of DAPK1 in poor-risk AML is enforced by a Flt3-to-NIK-to-p52NFkB2/relB signal cascade, and support a causative influence on clinical outcomes. Thus, resistance is subject to inactivation by a Flt3-selective TKI, in combination with HDAC inhibitor/or by proteasome-NFkB inhibitor with consequent ER stress apoptosis. A potential subsidiary role for syk inhibition downstream from Flt3 also exists. Taken together, these maneuvers reverse the resistance to apoptosis of NK Flt3-ITD or tMLL primary AML blasts, and including others with over-expressed/active Flt3 wild-type. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Nearly 24% of the world's population carry hemoglobin (Hb) gene variants, with the large majority of affected births occurring in low-income countries. The most prevalent structural Hb variants are the recessive β-globin gene mutations, βS or S, βC or C, and βE or E1. Hb S mutation is prevalent in sub-Saharan Africa and in Central India. Hb C is common in West Africa, and Hb E is common in Southeast Asia and in India. Homozygotes or compound heterozygotes with βS (e.g., Hb SS or SC) have sickle cell disease (SCD), a chronic sickling disorder associated with pain, chronic multi-organ damage, and high mortality. While Hb EE causes only a mild microcytic anemia, Hb E in combination with β-thalassemia can lead to transfusion dependent thalassemia. Though carriers are typically asymptomatic, they may pass the mutations to their offspring. Screening is needed so that these disorders can be diagnosed early and managed in a timely manner2. For example, in low-income countries, due to lack of nationwide screening and comprehensive care programs, up to 80% of babies born with SCD are undiagnosed and less than half of them survive beyond 5 years of age2. The unmet need for affordable, portable, accurate point-of-care tests to facilitate decentralized hemoglobin testing in resource-constrained countries is well-recognized 2,3. Here, we present international multi-site clinical validation results and high diagnostic accuracy of the 'HemeChip' (Fig. 1), an affordable, 10-minute point-of-care microchip electrophoresis test for identifying common Hb variants S, C, and E. Methods: Institutional Review Board approvals were obtained at each study site, and blood samples were collected as part of the standard clinical care. Tests were performed by local users, including healthcare workers and clinical laboratory personnel. 315 children (6 weeks to 5 years of age) were tested in Kano, Nigeria. Study participants were enrolled at three hospitals, Amino Kano Teaching Hospital, Murtala Mohammed Specialist Hospital, and Hasiya Bayero Pediatric Hospital. 124 subjects (7 weeks to 63 years old) were included in the study at Siriraj Thalassemia Center in Bangkok, Thailand. 298 subjects (8 months to 65 years old) were tested at a referral testing facility of ICMR-National Institute of Research in Tribal Health, located at Late Baliram Kashayap Memorial Medical College, Jagdalpur, Chhattisgarh, India. Blood samples were tested with both HemeChip and the standard reference methods, high performance liquid chromatography or cellulose acetate electrophoresis. Reference test results were not available to the HemeChip users. Similarly, HemeChip test results were not available to the users of the standard reference tests. Clinical validation studies presented here were performed with a fully functional, portable HemeChip prototype developed at Case Western Reserve University (Fig. 1A). A commercial product has been developed based on this technology by Hemex Health Inc. under the product name, GazelleTM(Fig. 1B). Results and Discussion: Among the total 768 tests performed with HemeChip in all test sites, 732 were valid tests, as defined by the Standards for Reporting Diagnostic Accuracy (STARD)4. HemeChip correctly identified all subjects with Hb SS, Hb SC, Hb AS, Hb AE, and Hb EE with 100% accuracy (Table 1). Nine subjects with normal Hb (Hb AA) were identified as HbSS in Nigeria. No subjects with disease were identified as normal or trait by HemeChip. Three subjects with compound heterozygous Hb Sβ-thalassemia (2 subjects with Hb Sβ+-thalassemia, 1 subject with Hb Sβ0-thalassemia) were identified as Hb SS. Sensitivity was 100% for all Hb types tested. Specificity was 98.7% for Hb SS versus other Hb types, and 100% for all other Hb types tested. HemeChip displayed an overall diagnostic accuracy of 98.4% in comparison to standard reference methods for the Hb variants tested in all clinical testing sites (Table 1). HemeChip is a versatile point-of-care system that enables affordable, accurate, decentralized hemoglobin testing in resource-limited settings. References: 1. Weatherall DJ, Clegg JB. Bull World Health Organ. 2001;79(8):704-712. 2. Mburu J, Odame I. International Journal of Laboratory Hematology. 2019;41(S1):82-88. 3. Alapan Y, Fraiwan A, Kucukal E, et al. Expert Review of Medical Devices. 2016;13(12):1073-1093. 4. Bossuyt PM, Reitsma JB, Bruns DE, et al. BMJ : British Medical Journal. 2015;351:h5527. Disclosures Fraiwan: Hemex Health, Inc.: Equity Ownership, Patents & Royalties. Hasan:Hemex Health, Inc.: Equity Ownership, Patents & Royalties. An:Hemex Health, Inc.: Patents & Royalties. Thota:Hemex Health, Inc.: Employment. Piccone:Hemex Health, Inc.: Patents & Royalties. Little:Hemex Health, Inc.: Patents & Royalties; GBT: Research Funding. Gurkan:Hemex Health, Inc.: Consultancy, Employment, Equity Ownership, Patents & Royalties, Research Funding.

Description: Parthenolide is a sesquiterpene lactone, a new class of antitumor agents which target nuclear factor kappa B (NFkB). Although the activation of NFkB has been implicated in the growth and drug resistance of multiple myeloma (MM) cells, the efficacy of parthenolide in MM is unknown. Here we show that parthenolide inhibits the NFkB DNA binding in MM cells in a time- and dose-dependent fashion. Parthenolide inhibits the proliferation of MM cell lines, as well as tumor cells from chemotherapy-resistant MM patients; however, at the effective doses it is not toxic to peripheral blood mononuclear cells or bone marrow stromal cells. Neither exogenous interleukin-6 (IL-6) nor insulin-like growth factor 1 (IGF-1) overcome the parthenolide-induced cytotoxicity. In addition, parthenolide inhibits the proliferation of MM cells adherent to bone marrow stromal cells. Parthenolide has a minimal effect on cell cycle progression but strongly induces apoptosis. Parthenolide activates caspases 8 and 3 more strongly than caspase 9, resulting in the cleavage of poly (ADP)-ribose polymerase (PARP) and XIAP. Z-VAD-FMK partially blunts the PARP cleavage but does not protect MM cells against apoptosis. Parthenolide induces p21 accumulation, while the levels of Bax, IAP2, cyclin D, Bcl-X and p53 or the phosphorylation status of JNK, AKT and ERK are unaffected. In addition, parthenolide augments dexamethasone-induced cytotoxicity. Our study therefore provides the rationale for the future clinical development of parthenolide, or its combination with dexamethasone, in MM therapy.