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Background: Over two thirds of patients with Multiple Myeloma (MM) are ≥ 65 years old at the time of diagnosis. Older adults are at greater risk for treatment related toxicity and inferior survival; such risks are inadequately explained by chronologic age and performance status. In 2015, the International Myeloma Working Group (IMWG) proposed a geriatric assessment (GA) based frailty index to identify older adults with MM at greatest risk of toxicities. Yet, routine implementation of GA in busy oncology practice remains challenging. We have previously shown the feasibility of a tablet-based modified Geriatric Assessment (mGA), capturing the Charlson Comorbidity Index (CCI), Katz Activity of Daily Living (ADL) Score, and Lawton Instrumental Activity of Daily Living (IADL) score, and its impact on clinical decision-making and treatment outcomes (Nathwani, et al. JOP. 2020). In the prior study, physicians recommended integration of the mGA into the electronic medical record (EMR) to improve usefulness at the clinic. Methods: We conducted a single institution pilot study to test the feasibility of integrating an electronic care planning system within the EMR such that with a single sign on, the dashboard showing results of the mGA was visualized within the EMR. Eligible patients had symptomatic MM, > 60 years old, and seeing their oncology providers to make a decision about treatment. After completing informed consent, patients completed a tablet-based mGA in clinic just prior to seeing the physician. Survey results were compiled and were immediately available for evaluation on a dashboard within the EMR. Providers reviewed the mGA results before meeting with the patient and completed a short survey after the visit regarding their own subjective impression of frailty and how the mGA influenced their treatment decision making. Agreement between provider's subjective vs mGA based frailty categorization was measured using Cohen's Kappa statistic. We measured relevant toxicity outcomes at 3 months post treatment initiation. Results: 25 patients were enrolled, with a median age of 68 (range=61-82), 52% (n=13) female, and 68% (n= 17) white. One patient did not complete the mGA survey and was not included in the analysis. The remaining patients completed the mGA successfully without interrupting clinic flow and mGA was immediately available for providers to review during the clinic visit. The average time providers spent reviewing results was of 5 (range 1-10) minutes. Providers subjectively categorized patients as 42% (n=10) fit, 58% (n=14) intermediate fit, and 0% (n=0) frail. According to the mGA, patients were 50% (n=12) fit, 29% (n=7) intermediate fit, and 21% (n=5) frail. There was an overall 46% (n=11) concordance between physician and mGA result. The most agreement was in fit status (58%, n=7) and least was frail (0%, n=0). There was 33% (n=4) agreement on intermediate fit status. The unweighted Cohen's kappa statistic was 0.09 indicating only slight agreement between the two methods. Providers reported mGA influenced their treatment decision in 33% (n=8), with the decision being either chemotherapy modification (n=6) or reduced dose transplant (n=2). One patient, who was frail and received induction treatment, died during the 3-month study period. The remaining patients (n=23) received treatment as planned. Discussion: In this study, we report the feasibility of an EMR integrated mGA tool completed by the patient prior to meeting with the physician. Patients completed the survey with assistance and without disrupting the clinic workflow. The mGA results were reviewed by providers in real time and influenced treatment decisions one third of the time. Nearly all patients (96%, n=24) completed therapy as planned. Providers tended to view the patients as more fit than the mGA result, suggesting that the mGA uncovers additional information related to the patient's ability to tolerate therapy. Toxicity follow up is ongoing and will be updated at the time of presentation. Disclosures Giri: CareVive: Honoraria, Research Funding; PackHealth: Research Funding. Costa: BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Speakers Bureau.

Background: Acute Myeloid Leukemia (AML) is a disease of older adults, with a median age of 68 years at diagnosis. The NCCN guidelines recommend comprehensive geriatric assessments (GA) be included in clinical practice to guide treatment decisions. Utility of GA in older AML patients in a real-world environment is not yet established. We tested the feasibility of using a modified GA (mGA), administered by patient self-report on a touchscreen computer, real-time use and utility by clinicians and the correlation of mGA results on treatment decision-making. Methods: 77 newly diagnosed patients were recruited from three sites to complete a tablet-based mGA screening at a treatment decision visit. The mGA includes four domains: age, activities of daily living (ADLs), instrumental ADLs, and comorbidities. Survey results along with history of falls was used to create the Frailty Index (FI). Providers were asked what they thought the fit/frailty status of the patient was before viewing the results on a dashboard. After viewing the survey results, the clinician discussed the treatment plan with the patient. Patients received intensive or non-intensive therapy. Additional information was captured on clinical trial enrollment. Baseline and 3 month surveys recorded presence and severity of 8 symptoms using the Edmonton Symptom Assessment Scale (ESAS) with 0=no symptom and 10=worst possible symptom and quality of life using the Functional Assessment of Cancer Treatment: Leukemia (FACT-LEU). Results: Participants had a median age of 71 years ( range:61-88y); 50% were female, and 87% white. Frailty Index results for 76 patients were 28 (36.4%) fit, 25 (32.5%) intermediate, and 23 (29.9%) frail. (One patient did not complete the mGA). 52 of 77 (69%) enrolled patients were alive at 3 months; 21(27%) died and 4 (5%) were lost to follow-up. Providers were asked the fit/frailty status prior to seeing the results of the mGA. Of 75 provider responses, results were 27 (36.0%) fit, 29 (38.7%) intermediate, and 19 (25.3%) frail. There was 63% (n=47) provider concordance with the mGA result. There was more agreement with fit (n=22, 81.5%) and frail status (n=11, 57.9%) and less with intermediate (n=14, 48.3%). Of the 25 of 75 (33.%) provider reports that indicated that the mGA result influenced the treatment decision, 6 patients (5 fit, 1 intermediate) received intensive treatment, 15 received non intensive treatment (1 fit, 6 intermediate, 8 frail) and 4 enrolled in a clinical trial (1 fit, 2 intermediate, and 1 frail). Significant symptom improvement at 3 months was seen for drowsiness, lack of appetite, shortness of breath, and anxiety. FACT Leu results did not change over 3 months. Providers reported an average of 4.45 minutes to review the dashboard. Patients were able to complete the surveys unassisted in an average time of 16.24 minutes. Discussion: There was nearly 40% discordance between the provider and mGA, with the most discordance on the intermediate fit status. However, results of the mGA influenced treatment decision making in one third of provider/patient interactions. Further analysis of the mGA domains is warranted to see if additional insights can be gained. With time, some symptoms improve and others don't. This points to the opportunity to direct resources towards symptom assessment during treatment. Feasibility was demonstrated in this study as providers received the aggregated results in real time and reviewed them in less than 5 minutes. In addition, patients were able to complete the survey unassisted without disturbing clinic operations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Human malaria is a complex disease and a leading cause of mortality in children under 5 years of age. Plasmodium falciparum (Pf) is the agent responsible for cerebral malaria. Parasite infected erythrocytes are sequestered in the brain vasculature, disrupting the blood-brain-barrier, and with systemic inflammation leading to progressive brain edema. The precise pathophysiologic mechanism(s) underlying brain swelling in CM is not known. Recent work from our laboratories indicates that there is a role for bradykinin (BK) in fluid transport in human brain microvascular endothelial cells (Front Med 6:75, 2019). We examined the role of bradykinin (BK) in pediatric CM. Initial studies showed recombinant falcipain-2, a cysteine protease contained in the parasite digestive vacuole, was inhibited by high molecular weight kininogen (HK), with an IC 50=36 nM. Further, falcipain-2, but not the related protease falcipain 3, hydrolyzed the chromogenic substrate S2302 (Pro-Phe-Arg-pNA) at pH 7.4 with an 88 nM K m. These results suggest that falcipain-2 has plasma kallikrein-like activity. HK is both an inhibitor and substrate of falcipain-2. Molar excess HK to falcipain-2 (ratio 8:1 to 2:1) blocked the proteolytic activity of the cysteine protease at pH 7.4. Equal molar falcipain-2 to HK (1:1) resulted in kallikrein-like cleavage of HK with stable BK liberation over 1 h. Molar excess falcipain-2 to HK (1:2 and greater) led to progressive HK cleavage into smaller proteins and peptides. The falcipain-2 major cleavages observed by N-terminal sequencing were in Domain 3 of the heavy chain of HK, the cysteine protease inhibitory region (I 292ASFSQNCDIYPGKDF 303, D 320IPTNSPELEETLT 334, and E 412KKIYPTVNCQPLG 425). P. falciparum trophozoite lysates completely hydrolyzed purified and plasma HK into a ~64 kDa heavy chain and ~46 kDa light chain in buffer containing EDTA, pepstatin, and PMSF. The cysteine proteinase inhibitor E64 blocked this cleavage, suggesting that the relevant activity was that of a cysteine protease. Plasma from Kenyan children presenting with CM (fever, parasitemia, coma) had evidence of circulating cHK, indicative of BK released from HK. Forty percent (8 of 20) of CM patients had no intact 120 kDa HK at hospital entry. In contrast, only 16% (3 of 8) of children with uncomplicated malaria had detectable cHK. In CM patients, the HK level before antimalarial treatment (58 ± 3.9 µg/ml) was significantly lower than the value after clinical recovery (69 ± 3.6 µg/ml; p These data strongly suggest that falcipain-2 liberates BK from HK by acting like plasma kallikrein and in high concentrations destroys HK's cysteine protease inhibitory region. Some children with CM have in vivo evidence of prior HK proteolysis. Total kininogen deficiency protects mice from lethal experimental CM. Taken together, these data suggest that bradykinin is a proximal mediator of cerebral malaria. Figure 1 Figure 1. Disclosures McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria.

It has long been recognized that individuals who express variations of the hemoglobin-A (HbA) protein experience less severe malaria disease. As malaria remains to be one of the most significant infectious diseases in history, this human adaptation has led to the persistence of HbA variants (HbVARs) in the population. The intricate lifecycle of the parasite which causes the most cases of clinical malaria, Plasmodium falciparum, relies on both asexual and sexual reproductive cycles, with host to vector transmission reliant on sexual stage gametocyte formation. Multiple epidemiological studies have shown that HbVARs may influence gametocyte production during P. falciparum infection, with greater gametocyte numbers reported in individuals with hemoglobin variant containing erythrocytes (Hb VAR-Ery) when compared to hemoglobin A containing erythrocytes (Hb A-Ery). Here we provide experimental support for these studies by showing significantly higher sexual differentiation rates among parasites grown in Hb S containing erythrocytes (Hb S-Ery) obtained from sickle cell patients than those differentiated in Hb A-Ery (p=0.038). Because the digestion of hemoglobin is such an integral part of the intraerythrocytic cycle, we then sought to determine whether there was a difference between the hydrolysis efficiencies of HbA and other hemoglobin variants (HbVAR). By using a prominent recombinant P. falciparum hemoglobinase we found the hydrolysis efficiency of HbA to be significantly (p=0.0058) more efficient after 24 hours compared to a HbVAR sample containing mixed amounts of HbA, HbF, and HbS. To further determine whether there is a link between hemoglobin digestion efficiency and sexual differentiation, we therapeutically inhibited the hemoglobin digestion and hemozoin formation process in a culture of P. falciparum using sub-optimal doses of chloroquine diphosphate. We found a significant difference (p Disclosures No relevant conflicts of interest to declare.

Background: Structural variants are key recurrent molecular features of myeloma (MM) with two types of complex rearrangement, chromoplexy and chromothripsis, having been described recently. The contribution of these to MM prognosis, rapid changes in clinical behavior and punctuated evolution is currently unknown as is the mechanism by which they deregulate gene function. Methods: We analyzed two sets of newly diagnosed MM data: 85 cases with phased whole genome sequencing; and 812 cases from CoMMpass where long-insert whole-genome sequencing was available. Patient derived xenografts from five MM cases were used to generate epigenetic maps for the histone marks, BRD4, MED1, H3K27Ac, H3K4me1, H3K4me3, H3K9me3, H3K36me3 and H3K27me3. Results: In the 10X data the median number of structural events per case was 25 (range 1 - 182); with a median of 14 intra-chromosomal events (range 1 - 179; P Using an elbow test defined cutoff, we identified cases with high structural variant load in 10% of cases. Chromoplexy called by "Chainfinder" was seen in 18% of cases. Chromothripsis called by "Shatterseek" was seen in 9% of cases. Cases with a high structural load alone were not associated with an adverse outcome whereas cases with chromoplexy or chromothripsis were associated with adverse PFS and OS, p=0.001. A new high-risk subgroup comprising approximately 5% of cases was identified with chromoplexy, chromothripsis and a high structural load. Gene set enrichment analysis of cases with chromoplexy and chromothripsis showed an excess of MYC, E2F and G2M targets, and a reduction in RAS signaling. Interferon a and g responses, an excess of TP53 and reduction in TRAF3 mutations was associated predominantly with chromothripsis. How chromoplexy and chromothripsis are tolerated by the cell is unknown and the association with the cGAS/STING response is further being explored. To determine how chromoplexy may deregulate multiple genes we identified the full spectrum of structural variants to the immunoglobulin (Ig) and non-Ig loci. A range of genes are deregulated by Ig loci including MAP3K14 at a frequency of 2% confirming the importance of non-canonical NFkB signaling. A novel intra-chromosomal rearrangement to ZFP36L1 was upregulated in 10% of cases but was not prognostic. Gene upregulation by non-Ig super enhancers is frequent and targets include PAX5, GLI3, CD40, NFKB1, MAP3K14, LRRC37A, LIPG, PHLDA3, ZNF267, CENPF, SLC44A2, MIER1, SOX30, TMEM258, PPIL1, and BUB3. The topologically associating domain (TADs) containing super enhancers bringing about gene deregulation include TXNDC5, FOXO3, FCHSD2, SP2, FAM46C, CACNA1C, TLCD2 and PIK3C2G. These super enhancers frequently contain important MM genes, the coding sequence of which are disrupted by the rearrangement and could contribute to the clinical phenotype. Accurately reconstructing the structure of the complex rearrangements will allow us to identify the mechanism of gene deregulation and to distinguish between either gene stacking, receptor stacking or both. Conclusions: Upregulation of gene expression by super enhancer rearrangement is a major mechanism of gene deregulation in MM and complex structural events contribute significantly to adverse prognosis by a range of mechanisms as well as simple gene overexpression. Disclosures Boyle: Amgen, Abbvie, Janssen, Takeda, Celgene Corporation: Honoraria; Amgen, Janssen, Takeda, Celgene Corporation: Other: Travel expenses. Walker:Celgene: Research Funding. Thakurta:Celgene: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Davies:Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor; Janssen, Celgene: Other: Research Grant, Research Funding. Morgan:Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding.

Krüppel-like factor 5 (Klf5) encodes a zinc-finger transcription factor and has been reported to be a direct target of C/EBPα, a master transcription factor critical for formation of granulocyte-macrophage progenitors (GMP) and leukemic GMP (L-GMP). Using an in vivo hematopoietic-specific gene-ablation model we demonstrate that loss of Klf5 function leads to a progressive increase in peripheral white blood cells, associated with increasing splenomegaly. Long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitors (MPP) were all significantly reduced in Klf5Δ/Δ mice, and knockdown of KLF5 in human CD34+ cells suppressed colony forming potential. ST-HSC, MPP and total numbers of committed progenitors were increased in the spleen of Klf5Δ/Δ mice, and reduced β1- and β2-integrin expression on hematopoietic progenitors suggests that increased splenic hematopoiesis result from increased stem and progenitor mobilization. Klf5Δ/Δ mice show a significant reduction in the fraction of Gr1+Mac1+ cells (neutrophils) in peripheral blood and bone marrow, and increased frequency of eosinophils in the peripheral blood, bone marrow and lung. Thus, these studies demonstrate dual functions of Klf5 in regulating hematopoietic stem and progenitor proliferation and localization in the bone marrow, as well as lineage choice post-GMP, promoting increased neutrophil output at the expense of eosinophil production. Refereed/Peer-reviewed

Complex developmental processes such as hematopoiesis require a series of precise and coordinated changes in cellular identity to ensure blood homeostasis. Epigenetic mechanisms help drive changes in gene expression that accompany the transition from hematopoietic stem cells to terminally differentiated blood cells. Genome-wide profiling technologies now provide valuable glimpses of epigenetic changes that occur during normal hematopoiesis, and genetic mouse models developed to investigate the in vivo functions of chromatin-modifying enzymes clearly demonstrate significant roles for these enzymes during embryonic and adult hematopoiesis. Here, we will review the basic science aspects of chromatin modifications and the enzymes that add, remove, and interpret these epigenetic marks. This overview will provide a framework for understanding the roles that these molecules play during normal hematopoiesis. Moreover, many chromatin-modifying enzymes are involved in hematologic malignancies, underscoring the importance of establishing and maintaining appropriate chromatin modification patterns to normal hematology.

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138+ cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138+ primary samples, but spared normal CD138− and CD34+ cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G0/G1 arrest and increased apoptosis in all cell-cycle phases, including G0/G1. To determine whether this regimen is active against quiescent G0/G1 MM cells, cells were cultured in low-serum medium to enrich the G0/G1 population. G0/G1–enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G0/G1–enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G0) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst+), low pyronin Y (PY)–staining (2N Hst+/PY−) G0 population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.

Introduction: Multiple myeloma (MM) is characterized by the over-expression of D-cyclin genes and the expression is tightly linked to cytogenetic subgroups. For instance, overexpression of CCND1 in the t(11;14) and CCND3 in the t(6;14) is direct through IgH super-enhancer translocation, and CCND2 overexpression in t(4;14), t(14;16) and t(14;20) indirectly, presumably as a result of transcription factor or epigenetic regulation. In hyperdiploid MM, CCND1 is highly expressed in patients with trisomy of chr.11 and CCND2 in those without trisomy 11. Here, we combine DNA methylation with CTCF or super-enhancers (SE) binding and global histone profile to describe a novel perspective of epigenetic regulation of CCND1/2 expression in MM. Methods: Newly diagnosed MM patient CD138 sorted bone marrow plasma cells (PCs), consisted of those with a t(14;16) (n=17), t(14;20) (n=7), t(4;14) (n=9), t(11;14) (n=10) and hyperdiploidy (n=19, separated into D1-HRD (n=12) or D2-HRD (n=7), and were compared to PCs from healthy donors (n=4). A reduced representation bisulfite sequencing was performed to identify differentially methylated regions (DMRs). Histone modifications were determined using ChIP-seq in reference to KMS11 cells for t(4;14), U266 for t(11;14), and MM.1S for the MAF group patients. Additionally, enrichment of SEs in MM1.S and CTCF in Delta47 cells were determined to assess the collaborative epigenetic impact on altered CCND1/2 expression (FDR Results: In the t(4;14) subgroup, CCND2 DMRs (n=21) at the promoter expressed marginal differences ( CCND1 showed a reducing hypomethylation gradient (MDM: 12% to 0.05%, p1%, p Conclusion: The disparities in CCND1/2 expression among MM subgroups are not the singular consequence from epigenetic events but broadly depends on other genotypic features such as alterations in copy number or chromosome structure. For instance, CCND1 DNA-demethylation does not always correlate with expression in t(4;14), D2-HRD, or the MAF cluster, implying that the predominant effect is through juxtaposition of the IgH-SE next to CCND1 in the t(11;14). In contrast, suppression of CCND2 expression in t(11;14), compared to t(4;14)/MAF may be explained by the enrichment of inactivating H3K27me3 marks at the body of CCND2 in the U266 cells. In summary, DNA hypomethylation at promoters or gene bodies facilitate the formation of open chromatin to enhance interactions with SE/CTCF or histones to constitute a three-dimensional epigenetic regulatory network, which potentially influence gene expression and identify novel variants in signaling pathways in MM, as evidenced here with CCND1/2. Disclosures Roy Choudhury: University of Arkansas for Medical Sciences: Employment, Research Funding. Davies:MMRF: Honoraria; TRM Oncology: Honoraria; ASH: Honoraria; Abbvie: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria. Morgan:Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria.

By functioning as an enzyme cofactor, hemoglobin component and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we used CRISPR/Cas9 to delete intron 1 and 8 GATA motifs in Alas2, encoding the rate-limiting enzyme in heme biosynthesis, in erythroid precursor G1E-ER-GATA-1 cells (Tanimura et al., 2016, EMBO Rep.). These cells stably express a b-estradiol-activated allele encoding the estrogen receptor hormone binding domain fused to GATA-1. The GATA motif mutations abrogated GATA-1-mediated activation of Alas2 and decreased heme ~30 fold. We merged GATA-1/heme-regulated sectors of the proteome and transcriptome. This analysis revealed a GATA-1/heme circuit involving hemoglobin subunits, ubiquitination components, and proteins not implicated in erythrocyte biology, including the zinc exporter Slc30a1. Slc30a1 and the zinc importer Slc39a8 were the most highly expressed zinc transporters in G1E-ER-GATA-1 cells. Both were GATA-1-activated, and Slc30a1, but not Slc39a8, expression declined in heme-deficient Alas2-intronic mutant cells. We tested whether the zinc transporter genes Slc30a1 and Slc39a8 that share GATA-1 regulation and differ in heme regulation are controlled similarly in primary cells. Lineage-depleted hematopoietic precursors from murine fetal livers were cultured for 72 hours, and flow cytometry was used to isolate cells based on erythroid surface markers. Whereas Slc30a1 expression increased 5.7-fold upon differentiation, Slc39a8 increased 4.6-fold during the initial phase, but was downregulated 8.7-fold thereafter. To quantify intracellular zinc levels, we used the fluorescent zinc indicator FluoZin-3. The FluoZin-3 signal increased during early maturation and decreased thereafter, as predicted by the zinc transporter switch, in which expression of the zinc exporter and importer was sustained and decreased, respectively, during terminal differentiation. We demonstrated that the zinc transporter switch occurs in a primary human erythroblast culture system. Intracellular zinc rises during initial erythroid maturation, followed by a steep decline during terminal differentiation. The heme biosynthesis inhibitor succinylacetone reduced SLC30A1 expression 2.8-fold (p < 0.01), as well as other heme-dependent globin genes HBB and HBA1. These primary mouse and human cell studies revealed an evolutionarily conserved mechanism to differentially regulate zinc transporter genes during erythroid differentiation, culminating in a switch from importer and exporter expression to solely exporter expression. To test whether reconfiguring the mechanism governing intracellular zinc impacts differentiation and to elucidate Slc30a1 and Slc39a8 function, we developed multiple shRNAs and used them in loss-of-function studies with lineage-negative hematopoietic precursors from murine fetal livers (E14.5). Slc39a8 downregulation decreased intracellular zinc levels in immature erythroblasts. We also used the zinc chelator TPEN to reduce zinc in immature erythroblasts. TPEN reduced intracellular zinc and considerably decreased the live cell population (p < 0.001). Downregulating Slc30a1 increased intracellular zinc 2.2-fold and, strikingly, accelerated differentiation (p < 0.001). This analysis established a conserved paradigm in which a GATA-1/heme circuit controls trace metal transport machinery and trace metal levels as a mechanism governing cellular differentiation. Ongoing studies involve the development of innovative strategies to leverage this mechanism to enhance or suppress erythrocyte development with primary mouse and human systems. We are conducting multi-disciplinary metallomic analyses to test the hypothesis that the dynamic changes in zinc selectively commission and decommission zinc-binding proteins in erythroblasts. We anticipate that this approach can be readily extended to other sectors of the hematopoietic system and more broadly. Disclosures No relevant conflicts of interest to declare.

Interactions between histone deacetylase inhibitors (HDACIs) and the novel proteasome inhibitor carfilzomib (CFZ) were investigated in GC- and activated B-cell–like diffuse large B-cell lymphoma (ABC-DLBCL) cells. Coadministration of subtoxic or minimally toxic concentrations of CFZ) with marginally lethal concentrations of HDACIs (vorinostat, SNDX-275, or SBHA) synergistically increased mitochondrial injury, caspase activation, and apoptosis in both GC- and ABC-DLBCL cells. These events were associated with Jun NH2-terminal kinase (JNK) and p38MAPK activation, abrogation of HDACI-mediated nuclear factor-κB activation, AKT inactivation, Ku70 acetylation, and induction of γH2A.X. Genetic or pharmacologic JNK inhibition significantly diminished CFZ/vorinostat lethality. CFZ/vorinostat induced pronounced lethality in 3 primary DLBCL specimens but minimally affected normal CD34+ hematopoietic cells. Bortezomib-resistant GC (SUDHL16) and ABC (OCI-LY10) cells exhibited partial cross-resistance to CFZ. However, CFZ/vorinostat dramatically induced resistant cell apoptosis, accompanied by increased JNK activation and γH2A.X expression. Finally, subeffective vorinostat doses markedly increased CFZ-mediated tumor growth suppression and apoptosis in a murine xenograft OCI-LY10 model. These findings indicate that HDACIs increase CFZ activity in GC- and ABC-DLBCL cells sensitive or resistant to bortezomib through a JNK-dependent mechanism in association with DNA damage and inhibition of nuclear factor-κB activation. Together, they support further investigation of strategies combining CFZ and HDACIs in DLBCL.

Interactions between the dual Bcr/Abl and aurora kinase inhibitor MK-0457 and the histone deacetylase inhibitor vorinostat were examined in Bcr/Abl+ leukemia cells, including those resistant to imatinib mesylate (IM), particularly those with the T315I mutation. Coadministration of vorinostat dramatically increased MK-0457 lethality in K562 and LAMA84 cells. Notably, the MK-0457/vorinostat regimen was highly active against primary CD34+ chronic myelogenous leukemia (CML) cells and Ba/F3 cells bearing various Bcr/Abl mutations (ie, T315I, E255K, and M351T), as well as IM-resistant K562 cells exhibiting Bcr/Abl-independent, Lyn-dependent resistance. These events were associated with inactivation and down-regulation of wild-type (wt) and mutated Bcr/Abl (particularly T315I). Moreover, treatment with MK-0457 resulted in accumulation of cells with 4N or more DNA content, whereas coadministration of vorinostat markedly enhanced aurora kinase inhibition by MK-0457, and preferentially killed polyploid cells. Furthermore, vorinostat also interacted with a selective inhibitor of aurora kinase A and B to potentiate apoptosis without modifying Bcr/Abl activity. Finally, vorinostat markedly induced Bim expression, while blockade of Bim induction by siRNA dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. Together, these findings indicate that vorinostat strikingly increases MK-0457 activity against IM-sensitive and -resistant CML cells through inactivation of Bcr/Abl and aurora kinases, as well as by induction of Bim.

The role of Bim in synergistic interactions between UCN-01 and MEK1/2 inhibitors in human multiple myeloma cells was investigated. Exposure of U266 or RPMI8226 cells to UCN-01 resulted in ERK1/2 activation-associated BimEL phosphorylation/down-regulation, events abrogated by MEK1/2 inhibitors. Enforced activation of ERK1/2 by transfection with constitutively active MEK1 diminished the capacity of PD98059 but not PD184352 to block UCN-01–mediated BimEL phosphorylation and to potentiate apoptosis. Cotreatment with MEK1/2 inhibitors increased the association of BimEL with both Bcl-2 and Bcl-xL in UCN-01–treated cells, leading to Bax/Bak conformational change and Bax mitochondrial translocation. Down-regulation of BimEL by shRNA substantially diminished UCN-01/MEK inhibitor-mediated Bax/Bak activation and apoptosis. Furthermore, transfection of cells with S65A Bim, a mutant resistant to UCN-01–mediated phosphorylation, significantly sensitized cells to UCN-01 lethality. Conversely, ectopic expression of either Bcl-2 or Bcl-xL did not alter UCN-01/MEK1/2 inhibitor-mediated modifications in BimEL phosphorylation but largely prevented cell death. Finally, IL-6 or IGF-1 failed to prevent MEK1/2 inhibitors from blocking UCN-01–induced BimEL phosphorylation/degradation or cell death. Collectively, these findings argue that UCN-01–mediated ERK1/2 activation leads to BimEL phosphorylation/inactivation, resulting in cytoprotection, and that interference with these events by MEK1/2 inhibitors plays a critical role in synergistic induction of apoptosis by these agents.

Interactions between UCN-01 and HMG-CoA reductase inhibitors (ie, statins) have been examined in human leukemia and myeloma cells. Exposure of U937 and U266 cells to minimally toxic concentrations of UCN-01 and various statins (eg, lovastatin, simvastatin, or fluvastatin) dramatically increased mitochondrial dysfunction, caspase activation, and apoptosis. Comparable effects were observed in other leukemia and myeloma cell lines as well as in primary acute myeloid leukemia (AML) blasts but not in normal hematopoietic cells. Potentiation of UCN-01 lethality by lovastatin was associated with disruption of Ras prenylation and activation. These events were significantly attenuated by farnesyl pyrophosphate (FPP) but not by geranylgeranyl pyrophosphate (GGPP), implicating perturbations in farnesylation rather than geranylgeranylation in synergistic interactions. Coexposure to statins and UCN-01 resulted in inactivation of ERK1/2 and Akt, accompanied by JNK activation. U266 cells ectopically expressing JNK1-APF, a dominant negative JNK1 mutant, displayed significantly reduced susceptibility to lovastatin/UCN-01–mediated lethality. Moreover, transfection of U266 cells with constitutively activated H-Ras (Q61L) attenuated ERK1/2 inactivation and dramatically diminished the lethality of this regimen. Collectively, these findings indicate that HMG-CoA reductase inhibitors act through a Ras farnesylation-associated mechanism to induce signaling perturbations, particularly prevention of Ras and ERK1/2 activation, in UCN-01–treated cells, resulting in the synergistic induction of cell death.

Interactions between the tyrphostin adaphostin and proteasome inhibitors (eg, MG-132 and bortezomib) were examined in multiple human leukemia cell lines and primary acute myeloid leukemia (AML) specimens. Cotreatment of Jurkat cells with marginally toxic concentrations of adaphostin and proteasome inhibitors synergistically potentiated mitochondrial damage (eg, cytochrome c release), caspase activation, and apoptosis. Similar interactions occurred in other human leukemia cell types (eg, U937, HL-60, Raji). These interactions were associated with a marked increase in oxidative damage (eg, ROS generation), down-regulation of the Raf/MEK/ERK pathway, and JNK activation. Adaphostin/MG-132 lethality as well as mitochondrial damage, down-regulation of Raf/MEK/ERK, and activation of JNK were attenuated by the free-radical scavenger NAC, suggesting that oxidative damage plays a functional role in antileukemic effects. Ectopic expression of Raf-1 or constitutively active MEK/ERK or genetic interruption of the JNK pathway significantly diminished adaphostin/MG-132-mediated lethality. Interestingly, enforced Raf or MEK/ERK activation partially diminished adaphostin/MG-132-mediated ROS generation, suggesting the existence of an amplification loop. Finally, the adaphostin/MG-132 regimen displayed similar toxicity toward 5 primary AML samples but not normal hematopoietic progenitors (eg, bone marrow CD34+ cells). Collectively, these findings suggest that potentiating oxidative damage by combining adaphostin with proteasome inhibitors warrants attention as an antileukemic strategy.

The transcription repressor BCL-6 is known to play critical roles in B-cell lymphomagenesis, germinal center formation, and balanced Th1/Th2 differentiation. In macrophages, although BCL-6 has also been shown to regulate the expression of several chemokine genes, its function in other aspects of macrophage biology has not been studied. In addition, the precise role of BCL-6 in cell proliferation is poorly understood in general. Here we report that BCL-6-/- macrophages hyperproliferate due to an accelerated G1/S transition accompanied by increased cyclin D2 and c-myc and decreased expression of p27. Crucial to this enhanced proliferation is spontaneous interleukin 6 (IL-6) production and signal transducer and activator of transcription 3 (STAT3) activation in BCL-6-/- macrophages. In colony-forming assays, BCL- 6-/- bone marrow progenitor cells form spontaneous macrophage colonies that can be inhibited by anti-IL-6 antibodies. Gene expression studies demonstrate that BCL-6 binds to several sequence motifs scattered in the IL-6 locus and can repress IL-6 transcription both in 293T cells and in macrophages. In conclusion, our results indicate that BCL-6 negatively regulates proliferation of the monocytic/macrophage lineage by suppressing an autocrine IL-6/STAT3-mediated gene expression program. Our work also suggests that BCL-6 prevents abnormal Th2 differentiation by suppressing basal level IL-6 production in antigen-presenting cells (APCs). (Blood. 2005;105:1777-1784)

Interactions between the cyclin-dependent kinase (CDK) inhibitor flavopiridol and the proteasome inhibitor bortezomib were examined in Bcr/Abl+ human leukemia cells. Coexposure of K562 or LAMA84 cells to subtoxic concentration of flavopiridol (150-200 nM) and bortezomib (5-8 nM) resulted in a synergistic increase in mitochondrial dysfunction and apoptosis. These events were associated with a marked diminution in nuclear factor κB (NF-κB)/DNA binding activity; enhanced phosphorylation of SEK1/MKK4 (stress-activated protein kinase/extracellular signal-related kinase 1/mitogen-activated protein kinase kinase 4), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK); down-regulation of Bcr/Abl; and a marked reduction in signal transducer and activator of transcription 3 (STAT3) and STAT5 activity. In imatinib mesylate-resistant K562 cells displaying increased Bcr/Abl expression, bortezomib/flavopiridol treatment markedly increased apoptosis in association with down-regulation of Bcr/Abl and BclxL, and diminished phosphorylation of Lyn, Hck, CrkL, and Akt. Parallel studies were performed in imatinib mesylate-resistant LAMA84 cells exhibiting reduced expression of Bcr/Abl but a marked increase in expression/activation of Lyn and Hck. Flavopiridol/bortezomib effectively induced apoptosis in these cells in association with Lyn and Hck inactivation. The capacity of flavopiridol to promote bortezomib-mediated Bcr/Abl down-regulation and apoptosis was mimicked by the positive transcription elongation factor-b (P-TEFb) inhibitor DRB (5,6-dichloro 1-β-d-ribofuranosylbenzinida-sole). Finally, the bortezomib/flavopiridol regimen also potently induced apoptosis in Bcr/Abl- human leukemia cells. Collectively, these findings suggest that a strategy combining flavopiridol and bortezomib warrants further examination in chronic myelogenous leukemia and related hematologic malignancies. (Blood. 2004;104:509-518)