ALBERT

Description: The erythroid Kruppel-like factor, EKLF, is an essential transcription factor for mammalian β-type globin gene switching, and specifically activates transcription of the adult β-globin gene through binding of its zinc finger domain to the β-globin promoter. We report now that EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives. We found that specific knockdown of EKLF levels by siRNA prevents SCFA induced-expression of an integrated γ-globin promoter in a stably-expressed mLCRβprRluc AγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. In chromatin immunoprecipitation (ChIP) assays, EKLF was found to be actively recruited to the endogenous γ-globin gene promoter with exposure of human erythroid progenitors, and hematopoietic cell lines, to SCFA derivatives. The human SWI/WNF complex is a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure in an ATP-dependent manner. We found that the SWI/SNF complex chromatin-modifying core ATPase BRG1 is also required for γ-globin gene induction by SCFA derivatives. Furthermore, BRG1 is actively recruited to the endogenous γ-globin promoter of human erythroid progenitors with exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings all demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. Recently. we also identified a γ-globin-specific repressor complex, consisting of NCoR and HDAC3, which is displaced from the proximal γ-globin promoter by exposure to SCFA derivatives prior to activation of transcription (Blood, 108:3179–86, 2006). Collectively, these studies identify critical activating and repressing cofactors regulating γ-globin gene expression, and provide new targets for therapeutic interventions.

Description: The dual bromodomain Brd2 is closely related to the basal transcription factor TAFII250, which is essential for cyclin A transactivation and mammalian cell cycle progression. Constitutive expression of BRD2 (under Eμ control) in the lymphoid lineage of transgenic mice elevates basal transcription of cyclin A, destabilizes the cell cycle and leads to B cell leukemias and lymphomas that are monoclonal, morphologically uniform, transplantable and highly malignant. The surface immunophenotype of the lymphoma cells is: B220+, CD19+, sIgM+, CD5+, CD9+; B7-1 and B7-2 elevated, CD23low; CD11b-, Ly-6G- and CD49b-, supporting lymphoid-restricted lineage; CD117- (c-kit) and CD127- (interleukin-7 receptor α-chain), consistent with mature cells; CD25-, syndecan-1-, and CD69- and exhibit a high IgM/low IgD ratio, which taken together identify a B-1 cell type. Malignant, proliferating cells infiltrate lymph nodes, liver, lung and kidney, and at late stages, cause anemia and a fatal peripheral leukemia over a 14-day time course from tumor cell transplantation to death. Genome-wide transcriptional expression profiling of these lymphoma cells reveals a transcriptional fingerprint that is most similar to human diffuse large B cell lymphoma (DLCL) with an “activated B cell” signature, consistent with histopathology, and establishes a novel murine DLCL model. DLCL is the most common type of non-Hodgkin’s lymphoma (NHL) in humans; all lymphomas combined are the fifth most common type of cancer diagnosed and the sixth most common cause of death in the United States. We treated syngeneic transplanted mice with CHOP (i.e. cyclophosphamide 40 mg/kg i.v., doxorubicin 3.3 mg/kg i.v., vincristine 0.5 mg/kg i.v. and predisone 0.2 mg/kg p.o. every day for 14 days) and monitored individual lymphoma cells, tagged with human-CD4+, by flow cytometry of lymphoid and non-lymphoid organs. In a novel approach, we supplemented CHOP with DNA oligonucleotides that mimic the chromosomal telomere, which we call a “T-oligo.” Normal cells exposed to this drug in vitro undergo transient cell cycle arrest, but DLCL cells undergo p53-dependent apoptosis. The mechanism immediately suggests a novel method of chemotherapy for leukemia and lymphoma to supplement CHOP. In mice treated systemically with CHOP and T-oligo, we observed major reductions in the leukemic burden in peripheral blood, reduced lymphadenopathy, reduced leukemic infiltrates of non-lymphoid organs and splenomegaly in the combined (“CHOP+T”) regimen over CHOP alone. We also confirmed in normal B lymphocytes that T-oligo causes only cell cycle arrest, not apoptosis. Mice likewise showed low toxicity to T-oligo at the effective doses, opening the way to a more extensive pre-clinical trial of this novel approach to NHL therapy.

Description: High-potency, non-cytotoxic oral therapeutics to stimulate both fetal globin expression and erythropoiesis are needed for optimal definitive therapy of the beta-hemoglobinopathies and thalassemias. Butyrate and phenylbutyrate have been the most active of the non-chemotherapy inducing agents, but their efficacy is limited by anti-proliferative effects. A discovery program to identify more potent fetal-globin inducing compounds has been pursued utilizing pharmacophore and pseudoreceptor models to screen chemical libraries for additional inducing agents, based initially on short chain fatty acid derivatives. From a library of 10,000 compounds, our pseudoreceptor model predicted that 30 candidate compounds would bind a structural receptor which results in induction of the γ globin gene promoter. Of the 30 candidates, two compounds (RB 4 and 9) were found to have inducing activity comparable to butyrate in reporter gene assays (producing 2-fold induction), two candidates (RB7 and 29) had higher potency (3–4 fold induction), and 9 compounds had intermediate (lower) inducing activity. Second, five compounds identified through the molecular modeling stimulated proliferation of cord blood erythroid progenitors significantly, even in the absence of any added cytokines. Four of the novel compounds induced fetal globin mRNA (assayed by RT-PCR) in erythroid progenitors by 1.5 to 7-fold over control cells, at concentrations one log lower than previously identified inducers, while butyrate induced γ mRNA by 2-fold but reduced erythroid colony growth by 15%. Of compounds studied in vivo, three candidate compounds induced γ globin mRNA (by 2 to 6-fold) over basal levels in baboons, or by 2–15-fold in mice transgenic for the human γ globin genes. Molecular mechanisms of fetal globin induction by the high-potency compound RB7 resulted from displacement of an HDAC3/NCoR repressor complex from the γ globin promoter, with concurrent local histone acetylation, binding of a remodeling complex, and recruitment of RNA Pol II. These studies together identify 1) novel therapeutic targets, and 2) therapeutic candidates with multiple beneficial actions for treating beta globin disorders and other anemias. Development of a high-potency, orally tolerable therapeutic to benefit even severely anemic thalassemia patients should now be feasible.

Description: High-level expression of fetal (Υ) globin reduces clinical complications in sickle cell disease and this is achieved with hydroxyurea (HU) in young children. However, non-cytotoxic high-potency therapeutics, particularly which can be utilized in combination with HU, are needed for most adolescent and adult patients who have continued serious clinical events. We have identified additional pharmaceutical candidates which induce HbF without cytotoxicity, using a Υ-globin gene promoter linked to GFP for robotic high-throughput screening, and screening five diverse chemical libraries. From a library of US and EU drugs which are approved for treatment of other medical conditions, a small panel of approved therapeutics were found to induce Υ globin expression, and have benign safety profiles, are orally active, and are suitable for long-term use. Three orally active candidates were evaluated in anemic baboons, and two, DLT and PB-04, induced Υ-globin mRNA by 15- to 33-fold over baseline levels. In 3/3 beta-globin locus YAC transgenic mice, one candidate (PB-04; 20 mg/kg) given by intra-peritoneal (IP) injections (for experimental feasibility) 3 times/ week for 5 wks significantly increased F-cells from 0.1 to 9%, 0.4 to 18%, and 0.13 to 12% respectively; and mean fluorescence intensity (MFI) increased by 10- to 33-fold. Responses were observed within one week. In hydroxyurea treated mice (100 mg/kg; IP, 5 days/ wk) F-cells increased from 0.3 to 2.3% on average (p

Description: Development of non-cytotoxic, orally-active therapeutics, which induce fetal globin production, would be beneficial for treatment of beta-thalassemias and hemoglobinopathies. The short-chain fatty acids (SCFA) butyrate and phenylbutyrate are active fetal globin inducers, but their clinical application has been limited by inhibition of erythropoiesis and unfavorable PK profiles. HQK-1001 is a SCFA which stimulates fetal globin gene expression in reporter gene assays, erythroid progenitors, transgenic mice, and anemic baboons. HQK-1001 also stimulates human erythroid progenitor (BFU-E) proliferation by a mean of 30% above controls, and increased red blood cell counts in phlebotomized baboons. HQK-1001 has demonstrated a wide safety margin in pre-clinical toxicology testing, with no significant adverse effects in sub-chronic (6-month) studies and negative mutagenicity testing. Based on these pre-clinical data, a placebo-controlled, randomized, blinded, single-dose escalating Phase 1 trial was conducted to evaluate HQK-1001 at 4 dose levels ranging from 2 to 20 mg/kg in 32 normal volunteers. The drug was welltolerated, with no significant adverse events. Pharmacokinetic analyses showed low intersubject variability, a linear increase in Cmax and AUC with increasing dose, and T½ of 11 hours, indicating suitability for once-daily dosing. Administration under fed conditions demonstrated equivalent AUC to fasting conditions. In a subsequent 14-day Phase 1 dosing study in 41 normal subjects, the drug was also well-tolerated, with no significant adverse effects at the studied dose levels (5, 10 and 15 mg/kg). F-reticulocytes increased in subjects treated with 5 to 10 mg/kg doses. Absolute reticulocytes increased significantly above baseline (p

Description: Key Points RP2D of PEV 20 mg/m2 in PEV/AZA combo did not alter toxicity profile of AZA; dose-limiting toxicities were transiently elevated AST/ALT. In treatment-naive older AML patients, the intent-to-treat ORR was 50%.

Description: Orally bioactive compounds that induce γ globin gene expression at tolerable doses are needed for optimal treatment of the β-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce γ globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce γ globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which γ globin can be induced. Selected short-chain fatty acid derivatives (SCFADs) were recently found to induce γ globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human β globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, γ globin mRNA increased 2-fold, reticulocytes increased 3- to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2- to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate γ globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the β hemoglobinopathies and useful for the oral treatment of other anemias.

Description: The mechanisms by which pharmacologic agents stimulate γ-globin gene expression in β-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with β-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of γ-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in γ-globin protein synthetic levels above baseline γ globin ratios and a relative decrease in β-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the γ-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, CP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which γ-globin gene expression is being altered strongly suggest that nuclear protein binding, including CP2, to the BRE-G1 region of the γ-globin gene promoter mediates butyrate activity on γ-globin gene expression. © 1998 by The American Society of Hematology.

Description: Abstract 977 Several classes of HbF inducers, including chemotherapeutic agents, ESAs, and short chain fatty acids (SCFADs) have demonstrated efficacy in inducing HbF in beta hemoglobinopathy and thalassemia patients, but broad clinical application of agents except Hydroxyurea (HU) has been limited by requirements for parenteral administration, high doses and toxicity concerns for long-term use. Yet, combination therapy is likely necessary to achieve the high fetal globin expression levels necessary to ameliorate anemia in the severe beta thalassemias. HQK-1001 is an oral, non-mutagenic SCFAD, which induces fetal globin gene expression through displacement of a repressor complex from the gamma globin gene promoter, and has shown a favorable safety and PK profile with a T1/2 of 11 hours with once daily oral doses from 5-20 mg/kg in normal human volunteers. To investigate whether HQK-1001 has additive activity with other fetal globin inducing agents with different mechanisms of action, dose-escalation studies of HQK-1001 were performed in baboons, and PK profiles and fetal globin mRNA were compared to treatment with HU. Fetal globin mRNA increased with escalating doses of HQK-1001 from 50-150 mg/kg, reaching peak effects at an AUClast of 1267 h.micrograms/ml, comparable to a human equivalent dose of 20 mg/kg (AUClast = 1174-2205 h. microgram/ml), while a significantly higher AUClast (11,817 h.microgram/ml) was observed at a 200 mg/kg/dose and associated with gamma globin suppression. In baboons treated with sequentially administered Hydroxyurea (HU) (3 days/week), gamma globin mRNA increased by 80-120% above baseline, treatment with HQK-1001 increased γ globin mRNA by 290% above baseline, while the 2-agent combination increased γ globin mRNA by 360%. In Bfu-e cultures comparing HQK-1001 +/− decitabine, F-cells increased above baseline in cultures treated with HQK-1001 alone by 32-71%, with decitabine alone by 66-104%, and with the 2 agents together by 147% above F-cell levels in control cultures. In cultures containing SCF, Bfu-e numbers increased by 25% over controls with both agents added to a peak decitabine conc. of 0.1 microM; erythroid growth declined at higher concentrations of decitabine. These studies together demonstrate higher gamma globin gene induction with HQK-1001 in combination with HU and with decitabine, and with reduced cytotoxicity with added HQK-1001. Dose-escalating Phase 2 clinical trials of HQK-1001 have begun in patients with beta thalassemia. Disclosures: Perrine: HemaQuest Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Faller:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Boosalis:HemaQuest Pharmaceuticals: Equity Ownership. Thomson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Berenson:hemaQuest Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Background Systemic AL amyloidosis is a clonal plasma cell disorder in which amyloid fibrils are deposited in tissues and organs, leading to multi-system organ dysfunction. The most frequently involved organs are the heart and kidney (individually or together), with advanced cardiac involvement conferring particularly poor outcomes. Achievement of hematologic response and improved organ function result in better outcomes. There are currently no approved treatments for AL amyloidosis; multiple myeloma (MM) treatment strategies are used for these pts. Active, tolerable treatment options specific for AL amyloidosis are needed. The oral proteasome inhibitor (PI) ixazomib (ixa) is active and approved in combination with lenalidomide (L)-dexamethasone (Dex) for the treatment of MM pts who have received ≥1 prior therapy. Methods RRAL pts with measurable disease and major organ involvement (cardiac/renal) who required treatment after 1-2 prior therapies (and were not refractory to prior PI therapy) were randomized to ixa (4.0 mg, d 1, 8, 15) plus Dex (20 mg, d 1, 8, 15, 22) or physician's choice (Dex alone or plus melphalan [M], cyclophosphamide [C], thalidomide [T], or lenalidomide [L]) in 28-d cycles until disease progression or unacceptable toxicity (or best response plus 2 cycles or maximum 18 mos therapy/600 mg total dose for MDex). Randomization was stratified by cardiac risk stage, relapsed vs refractory disease, and prior PI exposure. Primary endpoints were 1) overall hematologic response rate (ORR) centrally adjudicated, and 2) death or vital organ deterioration at 2 yrs. Key secondary endpoints were overall survival (OS) and hematologic complete response (CR) rate; other secondary endpoints included hematologic/vital organ progression-free survival (PFS), time to vital organ deterioration or mortality, duration of hematologic response (DOR), and safety. Results 168 pts were randomized to ixa-Dex (n=85) or physician's choice (n=83; 47 LDex, 24 MDex, 10 CDex, 2 TDex); median age was 65 (range 38-84) vs 66 (33-82) yrs, 60% vs 55% were male, 56% vs 63% had cardiac and 66% vs 58% had renal involvement (33% vs 23% had both) at initial diagnosis (plus 9% vs 12% liver, 12% vs 18% gastrointestinal tract, and 11% vs 10% peripheral nerve involvement), 47% vs 47% had prior bortezomib, and 47% vs 37% had prior transplant. Median time since diagnosis was 14.7 vs 15.9 mos. Hematologic responses were seen in 45 (53%) vs 42 (51%) pts receiving ixa-Dex vs physician's choice (odds ratio 1.10 [95% CI 0.60-2.01], p=0.762). Higher CR rates were seen with ixa-Dex vs physician's choice (26% vs 18%). Overall survival, overall/hematologic/vital organ PFS, time to vital organ deterioration/death, DOR, time to treatment failure, and time to subsequent therapy data all favored pts treated with ixa-Dex vs physician's choice (Figure). Vital organ response rates were 36% in the ixa-Dex arm vs 11% with physician's choice (cardiac response rate: 18% vs 5%; renal response rate: 28% vs 7%). At data cut-off, pts had received a median treatment duration of 11.7 vs 4.9 mos with ixa-Dex vs physician's choice, and 21% vs 6% of pts remained on treatment. Grade ≥3 adverse events (AEs) were seen in 59% vs 56% of pts, including 33% vs 41% with drug-related grade ≥3 AEs, 45% vs 33% had serious AEs, 25% vs 20% had AEs resulting in discontinuation, and there were 6% vs 5% on-study deaths. AEs of clinical importance included diarrhea (34% vs 30%), rash (33% vs 20%), cardiac arrhythmias (25% vs 15%), nausea (24% vs 14%), pneumonia (22% vs 16%), and peripheral neuropathy (20% vs 15%). Common (≥5% overall) grade ≥3 AEs were fatigue (9% vs 9%), anemia (2% vs 10%), cardiac failure, dyspnea (each 6% vs 4%), peripheral edema, and pneumonia (each 5% vs 5%). Conclusions Treatment with ixa-Dex significantly prolonged duration of composite survival and vital organ function, PFS, and time to subsequent therapy vs physician's choice. Moreover, ixa-Dex resulted in an improved CR rate and DOR and, although the primary endpoint of hematologic response was not met, all clinically relevant time-to-event endpoint data favored ixa-Dex vs physician's choice. Ixa-Dex was generally well tolerated and associated with a doubling of treatment duration vs physician's choice; no new safety signals were seen. TOURMALINE-AL1 is the first phase 3 trial in RRAL to show significant outcome improvements, suggesting ixa-Dex represents a new option for RRAL pts, who have limited access to therapies. Disclosures Dispenzieri: Akcea: Consultancy; Intellia: Consultancy; Alnylam: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Pfizer: Research Funding; Janssen: Consultancy. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Wechalekar:Takeda: Honoraria; GSK: Honoraria; Celgene: Honoraria; Amgen: Research Funding; Janssen-Cilag: Honoraria. Schönland:Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Other: Travel Grant. Sanchorawala:Proclara: Consultancy, Honoraria; Caelum: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Prothena: Research Funding; Celgene: Research Funding; Takeda: Research Funding. Landau:Pfizer: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Prothena: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Suzuki:Takeda: Honoraria; BMS: Honoraria, Research Funding; Ono: Research Funding; Celgene: Honoraria; Janssen: Honoraria. Comenzo:Takeda: Research Funding; Caelum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Prothena Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Unum: Membership on an entity's Board of Directors or advisory committees, Research Funding; Myself: Patents & Royalties: Patent 9593332, Pending 20170008966. Berg:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd: Employment, Patents & Royalties. Liu:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Faller:Phoenicia Biosciences: Equity Ownership; Briacell Pharmaceuticals: Equity Ownership; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment; Viracta Pharmaceuticals: Equity Ownership; Boston University: Employment. Off Label Disclosure: Investigation of the oral proteasome inhibitor ixazomib in combination with dexamethasone versus physician's choice (of which there are no approved treatment options) for the treatment of relapsed/refractory primary systemic amyloidosis.