ALBERT

Description: Background: The combination of MPD and amyloidosis is rare, and this patient population's clinical outcome is not well studied. Methods: Pts with a MPD and amyloidosis were identified via an clinical note search engine that searches through the electronic medical records of patients seen at Mayo Clinic Rochester, Jacksonville, and Scottsdale between 1990 and 2016. Terms used included amyloid or amyloidosis, chronic myelogenous leukemia or CML, essential thrombocytopenia or ET, and polycythemia vera or PV. Demographic and clinical data were abstracted from the medical record. Pts with both disorders were analyzed and their mortality rates along with median time to death were calculated. Prevalences at the Mayo Clinic were calculated for the years 2014 and 2015. Results: Twenty-three pts diagnosed with both a MPD and amyloidosis were identified. Thirteen (56.5%) were male, 10 (43%) were female. Eleven (47.8%) were initially diagnosed at the Mayo Clinic. Types of amyloidosis were as follows: Eleven (47%) had immunoglobulin light-chain (AL), four had localized (17%), two (8.7%) had wild-type transthyretin (ATTR), one (4.3%) had mutant ATTR, and five (21.8%) were unknown. Types of MPD were as follows: Seven (30%) had polycythemia vera (PV), seven (30%) had chronic myelogenous leukemia (CML), five (22%) had myelofibrosis, and four (17%) had essential thrombocytosis (ET). Fifteen (65%) were initially diagnosed with a MPD. Median time to last follow-up from second diagnosis was 1.7 years, and median time to death following second diagnosis was 1.4 years. The mortality rate was 87% in the total population. The median time to death for AL and PV was 2.7 years, AL and CML 1.0 years, and AL and ET 1.17 years. Myelofibrosis did not occur with AL. The most common combination was AL and PV, which accounted for five (22%) of the cases. Treatment regimens for the patients with AL were varied. Multiple drugs were combined with dexamethasone including melphalan, velcade, pomalidomide, lenalidomide, doxorubicin, and revlimid. Cyclophosphamide, bortezomib, and dexamethasone (CyBorD) were used in two cases. The mean time to diagnosis of amyloidosis from symptom onset was 10.3 months. Prevalences of AL and a MPD in 2014 and 2015 at the Mayo Clinic were 2.8% and 1.5%, respectively. Conclusions: The mortality rate for the combined diagnoses is high. The most common combination of diagnoses was that of AL and PV, which was associated with a mean time to death of 2.7 years. Disclosures Al-Kali: Celgene: Research Funding; Onconova Therapeutics, Inc.: Research Funding.

Description: Introduction: A recent study revealed an antiproliferative and apoptotic effect of propranolol on multiple myeloma (MM) cells. Our previous small matched case-control study showed longer survival in patients with propranolol and other beta-blockers (BB) intake than those without. This larger scale study was conducted to confirm the positive association of BB and MM survival. Methods: We identified 1971 newly diagnosed pts seen at Mayo Clinic between 1995 and 2010. Cardiac medication usage after diagnosis of MM was extracted from patient records and categorized based on BB intake. Cause of death was collected with death due to MM as the primary interest event and death due to cardiac disease or other reasons as competing risk events. The primary outcomes were MM disease-specific survival (DSS) and overall survival (OS). Cumulative incidence functions and Kaplan-Meier method were used to estimate the 5-year cumulative incidence rate (CIR) of MM death and OS rate, respectively. DSS and OS were compared by Gray's test and log-rank test, respectively. Multivarable Cox proportional hazard models were used to estimate the adjusted cause-specific HR (HRCSadj.) and hazard ratio (HRadj.) for DSS and OS, respectively, adjusting for demographics, disease characteristics, diagnosis year, and various chemotherapies. Results: 930 (47.2%) of MM patients had no intake of any cardiac medications; 260 (13.2%) had BB only; 343 (17.4%) used both BB / non-BB cardiac medications; and 438 patients (22.2%) had non-BB cardiac drugs. Five-year CIR of MM death and OS rate were shown in table. Superior MM DSS was observed for BB only users, compared to patients without any cardiac drugs (HRCSadj., .53, 95% confidence interval [CI], .42-.67, padj.

Description: Background: New therapies for AML are urgently needed. IMGN779 is a novel CD33-targeting ADC that utilizes as the cytotoxic agent a DNA-alkylatingindolinobenzodiazepine, DGN462. Previous data have shown that IMGN779 exerts dose-dependent activity against human CD33+ AML cells in vitro and in vivo. We hypothesized that combination treatment of AML cells with the poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, which blocks cellular DNA repair would further enhance the anti-leukemic activity of IMGN779 in preclinical human AML models. Methods:Human CD33+ AML cell lines (HEL, MV4-11, HL60) were treated in vitro with controls, IMGN779, olaparib, and IMGN779 + olaparib. Proliferation was measured by WST-8 reagent. Synergistic/additive effects were calculated using Compusyn software. Flow cytometry was performed to assess apoptosis, viability, and cell cycle effects. SCID mice were engrafted systemically with human AML (HEL-luciferase) cells followed by treatment with IMGN779, olaparib, or both drugs in combination. Changes in disease burden and possible treatment-related toxicities were determined by whole animal bioluminescent imaging, body weights, and time to morbidity, respectively. Primary cells from patients with relapsed/refractory AML characterized by complex karyotype or FLT-3 mutations were plated in methocellulose media supplemented with hematopoietic cytokines for colony formation unit assays (CFU). Vehicle, IMGN779 and/or olaparib in triplicate were added followed by quantification of leukemic CFU 15 days later with a Spot-RT3 camera mounted to an inverted microscope. Results: IMGN779 treatment induced significant growth inhibition in vitro in all CD33+ human AML cell lines tested that was dose dependent. IMGN779 cell killing was CD33 dependent. Olaparib induced cell death in human AML cell lines via reversal of DNA damage repair mechanisms. Combination treatment with IMGN779 (500 pM-1 nM) and olaparib (10-50 μM) significantly enhanced anti-leukemic effects over monotherapy in the same cell lines (representative data in Table 1). Combination indices for IMGN779 and olaparib therapy ranged from 0.7-0.9, consistent with synergistic effects. The combination markedly reduced overall cell viability, increased apoptosis, and induced almost complete S-phase cell cycle arrest as compared with controls and single-agent treatments. Exposure to a combination of IMGN779 and olaparib also significantly inhibited CFU growth of progenitor cells established from bone marrow samples of patients (n=7) with relapsed/refractory, FLT3-ITD, and/or complex karyotype AML. Statistically significant inhibition of viable CFUs was observed following combination IMGN779 (10 pM) and olaparib (1 μM) therapy as compared with monotherapy or vehicle controls (p

Description: Background. AML blasts from different patients vary in the agents to which they are most responsive. With a plethora of novel agents to evaluate, there is a lack of predictive biomarkers to precisely assign targeted therapies to individual patients. Primary AML cells often survive poorly in vitro, thus confounding conventional cytotoxicity assays. Dynamic profiling, i.e. functional biomarker assays of responsiveness to chemotherapeutic agents, is an alternative approach to help further the move towards personalised therapy. The purpose of this work was to assess the potential of two same-day dynamic profiling assays in AML cell lines to predict response to chemotherapy. (i) Ribosomal protein S6 (rpS6) is a downstream substrate of PI3K/akt/mTOR/ p70S6 kinase and MAPK/p90S6 kinase pathways and is also dephosphorylated following DNA double strand breaks. It thus has the potential to function as a biomarker of responsiveness to several therapeutic agents. (ii) Cellular propensity for apoptosis can be interrogated via a functional assay termed BH3 profiling. Dynamic BH3 profiling can be used to predict cellular responses to therapy based on priming mitochondria with BH3 domain peptides and thus allowing early detection of pro-apoptotic drug effects on mitochondrial outer membrane permeabilisation. Methods.We measured effects of a short (four hour) incubation with drugs of interest on rpS6 phosphorylation and BH3 peptide-accelerated cytochrome C release by flow cytometry. Baseline rpS6 phosphorylation was determined by culture with the mTOR inhibitor Rapamycin and the MEK inhibitor U0126. BH3 profiling included permeabilisation with digitonin followed by mitochondrial exposure to PUMA BH3-derived peptide. To establish specificity for sensitive cells we also measured dose responses to the drugs in 12 diverse AML cell lines after 48 hours' culture. Results. RpS6 dephosphorylation at four hours closely predicted the 48 hour IC50 for FLT3 inhibitors, an hsp90 inhibitor and topoisomerase II inhibitors. ROC (predictive test) analysis of small molecule inhibitors showed that the assay was highly sensitive and specific with area under the curve (AUC) values of 1.0 (sorafenib), 1.0 (AC220) and 1.0 (17-AAG). RpS6 phosphorylation also predicted response to the double strand break inducing drugs, with AUC values of 1.0 (mylotarg), 0.83 (etoposide) and 0.82 (vosaroxin). In contrast, responses to cytarabine and ABT-199, likely independent of mTOR, MAPK or double strand break response pathways, are not predicted by rpS6 dephosphorylation. PUMA-BH3 peptide-induced cytochrome C release also closely predicted the 48 hour IC50 with AUC values for FLT3 inhibitors of 1.0 (sorafenib) and 1.0 (AC220), double strand break inducing drugs 1.0 (mylotarg), 1.0 (etoposide) and 1.0 (vosaroxin) and a Bcl-2 targeting agent 0.875 (ABT-199). Response to 17-AAG and cytarabine were not predicted by this assay. Preliminary analysis shows that differential responses within primary AML sample subsets can be interrogated with these methodologies. Conclusions. In conclusion, we have established that rpS6 dephosphorylation and/or PUMA-BH3 peptide-induced cytochrome C release predict chemoresponsiveness to tyrosine kinase inhibitors, topoisomerase II inhibitors, an hsp90 inhibitor and a Bcl-2 targeting agent in AML cell lines after short term culture. Both assays are sensitive, specific and amenable to leukaemic sub-population analysis and are thus suitable assays for further development towards use in a clinical setting. pRS6 can be performed on smaller samples and is less technically challenging than priming with PUMA-BH3 peptide, but does not take into account apoptosis resistance that may occur independently of pathways effecting pRS6 inhibition. Disclosures No relevant conflicts of interest to declare.

Description: The t(8;21) chromosomal translocation is among the most frequent recurring cytogenetic abnormalities associated with acute myeloid leukemia (AML), found in 8-12% of de novo AML patients. The t(8;21) results in the stable fusion of the RUNX1 and RUNX1T1 genes, and formation of the oncofusion protein RUNX1-ETO (AML1-ETO). RUNX1-ETO is composed of the N-terminal DNA-binding domain of RUNX1 and nearly the entire ETO protein. RUNX1-ETO promotes leukemia development via the recruitment of transcription factor/transcriptional repression complexes (including NCOR, HDACs, p300, etc.) to regulatory regions of RUNX1 target genes known to be critical for myeloid differentiation and function, such as CEBPA, SPI1 (PU.1), NFE2, and CSF1R. Despite this knowledge, additional RUNX1-ETO target genes remain poorly characterized, and the complete molecular mechanism through which RUNX1-ETO leads to leukemic transformation remains to be elucidated. We propose that a better understanding of additional RUNX1-ETO target genes will lead to the potential for development of novel therapeutics to treat these patients. One such gene that we initially identified as markedly downregulated in RUNX1-ETO leukemia cells using a mouse model of t(8;21) AML is RASSF2 (Lo et al, Blood, 2012). Assessment of publicly available gene expression data revealed that RASSF2 is specifically downregulated in the bone marrow of t(8;21) AML patients compared to patients of different cytogenetic subtypes or to non-t(8;21) FAB subtype M2 AML patients. Additionally, RT-qPCR analysis confirmed that RASSF2 transcript is downregulated 10-100-fold in the t(8;21) AML cell lines, Kasumi-1 and SKNO-1, compared to non-t(8;21) AML cell lines and normal CD34+ hematopoietic cells. Expression of RUNX1-ETO in a non-t(8;21) AML cell line led to a reduction in RASSF2 mRNA expression, while knockdown of RUNX1-ETO in Kasumi-1 cells resulted in a ~5-fold increase in RASSF2 expression. Assessment of published ChIP-seq data showed that RUNX1-ETO directly binds at two regulatory regions within the RASSF2 genomic locus in t(8;21) AML cell lines and patient samples. Re-expression of RASSF2 at physiological levels in t(8;21) AML cell lines resulted in a modest negative growth phenotype, and greatly sensitized these cells to apoptosis following stimulation with various pro-apoptotic agents. Re-expression of RASSF2 in RUNX1-ETO-transduced primary mouse bone marrow caused these cells to lose their long-term self-renewal ability after 3 weeks in a serial replating/colony formation assay. This loss of self-renewal ability in co-transduced cells was accompanied by a marked increase in apoptosis during each of the first three weeks of replating. Mechanistically, re-expression of full-length RASSF2, but not of a deletion mutant lacking the SARAH heterodimerization domain (RASSF2ΔSARAH), in t(8;21) AML cell lines resulted in increased protein amount of the pro-apoptotic kinase, MST1. This suggests that RASSF2 may be a critical regulator of MST1 protein stability in AML cells. Importantly, modest (2-3-fold) overexpression of MST1 in t(8;21) AML cell lines resulted in a significant increase in apoptosis and caused growth arrest. The effects of RASSF2 or MST1 expression in non-t(8;21) AML cell lines were greatly reduced, suggesting that the cellular context of RUNX-ETO-driven leukemias makes them highly susceptible to MST1-dependent apoptosis. Overall, we have identified the importance of a MST1-driven pro-apoptotic signaling axis in t(8;21) leukemia. RUNX1-ETO-dependent transcriptional repression of RASSF2 may be essential for evasion of this apoptosis signaling during leukemic transformation via reduction of MST1 protein stability. MST1, perhaps better known as the mammalian orthologue of the drosophila Hippo kinase, is a critical tumor suppressor in many solid tumor types; and we believe our studies warrant the continued investigation of this pathway in hematological malignancy. Disclosures No relevant conflicts of interest to declare.

Description: Chemotherapy drugs tend to spare cells in a state of dormancy (G0 phase of the cell cycle). Relapse of acute myeloid leukaemia (AML) is likely in part due to dormant cells evading remission-induction chemotherapy. Dormant AML cells have been identified in the bone marrow endosteal region which is characterised by an excess of TGFβ1 and a shortage of nutrients. We developed and characterized an in-vitro model of AML cell dormancy by exploiting these features. Following preliminary investigation of several cell lines, the CD34+CD38- line TF1a was selected for in depth investigation. TF1a cells showed 72% inhibition of proliferation (p2 fold including genes involved in stemness, chemoresistance and tumour suppressor genes in addition to genes involved in canonical cell cycle regulation. There was striking upregulation of genes involved in adhesion and migration: raised expression levels of SPP1 (the gene coding for osteopontin), ITGB3, ITGB4, ITGA3 and CD44 in dormant cells were confirmed by real-time PCR. The most upregulated gene was SPP1/osteopontin (16 fold). Immunocytochemistry of biopsy material from AML patients confirmed high levels of osteopontin in the cytoplasm of blasts near the paratrabecular bone marrow. Osteopontin and other genes identified in this model, including well-characterised genes (e.g. CD44, CD47, CD123, ABBC3 and CDKN2B) as well as little-known ones (e.g. PTPRU, ITGB3 and BTG2), are potential therapeutic targets in dormant AML cells. Disclosures No relevant conflicts of interest to declare.