Human Hematological Cancer Research Articles

Insight into the Role of the miR-584 Family in Human Cancers.

Among the non-coding RNAs, the aberrant expression of microRNAs (miRNAs) is well described in the oncology field. It is clear that the altered expression of miRNAs is crucial for a variety of processes such as proliferation, apoptosis, motility, angiogenesis and metastasis insurgence. Considering these aspects, RNA-based therapies and the use of miRNAs as non-invasive biomarkers for early diagnosis are underlined as promising opportunities against cancer death. In the era of precision medicine, significant progress in next-generation sequencing (NGS) techniques has broadened knowledge regarding the miRNAs expression profile in cancer tissues and in the blood of cancer patients. In this scenario, pre-clinical and clinical studies suggested that the members of the miR-584 family, i.e., miR-584-5p and -3p, are prominent players in cancer development and progression. Under some conditions, these miRNAs are under-expressed in cancer tissues acting as tumor suppressors, while in other conditions, they are overexpressed, acting as oncogenes increasing the aggressive behavior of cancer cells. The aim of this review is to provide a comprehensive and up-to-date overview on the expression, upstream genes, molecular targets and signaling pathways influenced by the miR-584 family (i.e., miR-584-3p and -5p) in various human solid and hematological cancers. To achieve this goal, 64 articles on this topic are discussed. Among these articles, 55 are focused on miR-584-5p, and it is outlined how this miRNA could be used in future applications as a potential new therapeutic strategy and diagnostic tool.

Paracrine Factors Released from Tonsil-Derived Mesenchymal Stem Cells Inhibit Proliferation of Hematological Cancer Cells Under Hyperthermia in Co-culture Model.

Mesenchymal stem cells (MSCs) are promising biological therapeutic candidates in cancer treatment. As a source of MSCs, palatine tonsil tissue isone of the secondary lymphoid organs that form an essential part of the immune system, and the relation betweenthe secondary lymphoid organs and cancer progression leads us to investigate the effect of tonsil-derived MSCs (T-MSC) on cancer treatment. We aimed to determine the anti-tumoral effects of T-MSCs cultured at the febrile temperature (40°C) on hematological cancer cell lines. The co-culture of cancer cells with T-MSCs was carried out under fever and normal culture conditions, and then the cell viability was determined by cell counting. In addition, apoptosis rate and cell cycle arrest were determined by flow cytometry. We confirmed the apoptotic effect of T-MSC co-culture at the transcriptional level by using real-time polymerase chain reaction (RT-PCR). We found that co-culture of cancer cells with T-MSCs significantly decreased the viable cell number under the febrile and normal culture conditions. Besides, the T-MSC co-culture induced apoptosis on K562 and MOLT-4 cells and induced the cell cycle arrest at the G2/M phase on MOLT-4 cells. The apoptotic effect of T-MSC co-culture under febrile stimulation was confirmed at thetranscriptional level. Our study has highlighted the anti-tumoral effect of the cellular interaction between the T-MSCs and human hematological cancer cells during in vitro co-culture under hyperthermia.

Selective MCL-1 inhibitor ABBV-467 is efficacious in tumor models but is associated with cardiac troponin increases in patients

BackgroundMCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902).MethodsBinding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy.ResultsHere we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings.ConclusionsThe selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.

Development of Curcumin and Piperine-Loaded Bio-Active Self-Nanoemulsifying Drugs and Investigation of Their Bioactivity in Zebrafish Embryos and Human Hematological Cancer Cell Lines.

Curcumin (CUR) and piperine (PP) are bioactive compounds with prominent pharmacological activities that have been investigated for the treatment of various diseases. The aim of the present study is to develop Bio-SNEDDS for CUR and PP as a combined delivery system for cancer therapy. CUR and PP loaded Bio-SNEDDSs with varying compositions of bioactive lipid oils, surfactants, and cosolvents were prepared at room temperature. Bio-SNEDDSs were characterized using a Zetasizer Nano particle size analyzer and further examined by transmission electron microscopy (TEM) for morphology. The in vivo toxicity of the preparations of Bio-SNEDDS was investigated in wild-type zebrafish embryos and cytotoxicity in THP-1 (human leukemia monocytic cells), Jurkat (human T lymphocyte cells) and HUVEC (non-cancerous normal) cells. Bio-SNEDDSs were successfully developed with black seed oil, Imwitor 988, Transcutol P and Cremophor RH40 at a ratio of 20/20/10/50 (%w/w). The droplet size, polydispersity index and zeta potential of the optimized Bio-SNEDDS were found to be 42.13 nm, 0.59, and -19.30 mV, respectively. Bio-SNEDDS showed a spherical structure evident by TEM analysis. The results showed that Bio-SNEDDS did not induce toxicity in zebrafish embryos at concentrations between 0.40 and 30.00 μg/mL. In TG (fli1: EGFP) embryos treated with Bio-SNEDDS, there was no change in the blood vessel structure. The O-dianisidine staining of Bio-SNEDDS treated embryos at 48 h post-fertilization also showed a significant reduction in the number of blood cells compared to mock (DMSO 0.1% V/V) treated embryos. Bio-SNEDDS induced significant levels of cytotoxicity in the hematological cell lines THP-1 and Jurkat, while low toxicity in normal HUVEC cell lines was observed with IC50 values of 18.63±0.23 μg/mL, 26.03 ± 1.5 μg/mL and 17.52 ± 0.22 μg/mL, respectively. Bio-SNEDDS exhibited enhanced anticancer activity and could thus be an important new pharmaceutical formulation to treat leukemia.

Sensitivity of canine hematological cancers to BH3 mimetics.

Inhibition of antiapoptotic B-cell lymphoma 2 (BCL2) proteins by small molecule Bcl-2 homology 3 (BH3) mimetics causes rapid induction of apoptosis of human hematological cancers in vitro and in vivo. Assess in vitro sensitivity of non-neoplastic lymphocytes and primary hematological cancer cells from dogs to venetoclax (VEN) or the dual BCL2/ B-cell lymphoma-extra-large (BCLxL) inhibitor, navitoclax (NAV), and evaluate the association between BCL2 protein expression and VEN sensitivity. Nine client-owned dogs without cancer and 18 client-owned dogs with hematological cancer. Prospective, nonrandomized noncontrolled study. Lymphocytes isolated from peripheral blood, lymph node, or bone marrow from dogs were incubated with BH3 mimetics for 24 hours. Viable cells were counted using flow cytometry and half maximal effective concentration (EC50 ) was calculated. BCL2 protein from whole cell lysates was assessed via immunoblots. Nodal B and T lymphocytes were more sensitive to VEN than circulating lymphocytes (P=.02). Neoplastic T lymphocytes were sensitive to VEN (mean EC50 ± SD =0.023 ± 0.018 μM), whereas most non-indolent B cell cancers were resistant to killing by VEN (mean EC50 ± SD=288 ± 700 μM). Unclassified leukemias showed variable sensitivity to VEN (mean EC50 ± SD=0.49 ± 0.66 μM). Detection of BCL2 protein was not associated with VEN sensitivity. Neoplastic canine T lymphocytes are sensitive to VEN in vitro. Quantification of BCL2 protein alone is insufficient to predict sensitivity to VEN.

Phase 1b/2a Study of AZD4573 (CDK9i) and Acalabrutinib in Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma (r/r DLBCL): Results from Dose-Escalation

Background: Inhibition of Cyclin-dependent kinase 9 (CDK9), a transcriptional regulator, leads to short-term reduction of anti-apoptotic and oncogenic proteins. AZD4573, a potent and highly selective inhibitor of CDK9, triggers downregulation of MCL-1, BFL-1 and MYC, rapidly inducing apoptosis in human hematological cancer cell lines. The safety of AZD4573 as monotherapy was previously established in a Phase 1 trial in hematological malignancies (NCT03263637). Inhibition of Bruton tyrosine kinase with acalabrutinib increases expression of pro-apoptotic proteins such as Bim. Combining AZD4573 with acalabrutinib in DLBCL in in vitro models accelerates induction of cleaved Caspase-3, suggesting a synergistic mechanism that pushes malignant cells closer to their apoptotic threshold. Here, we report the dose-escalation of a multicenter, open-label, Phase 1b/2a study to assess safety and tolerability of AZD4573 with acalabrutinib in patients with r/r DLBCL (NCT04630756). Methods: Patients with r/r DLBCL with ≥2 prior lines of therapy (stem cell transplant or CAR-T allowed) were eligible. AZD4573 was given intravenously weekly and acalabrutinib 100 mg was given orally twice daily. In cycle 1, there was a weekly intrapatient dose ramp up of AZD4573 with a target dose of 9 mg in cohort 1 (3 mg, 6 mg, 9 mg) and 12 mg in cohort 2 (6 mg, 9 mg, 12 mg). Treatment was given until progressive disease/unacceptable toxicity. Responses were assessed by Lugano 2014 criteria and adverse events (AEs) were graded using CTCAE v5.0. The primary objective was to assess safety and determine the recommended phase 2 dose; pharmacokinetics and antitumor efficacy were secondary and exploratory endpoints, respectively. Results: As of August 2, 2022, 21 patients have been dosed. At data cutoff (DCO) on May 17, 2022, 13 patients were evaluable for safety (cohort 1, n=6; cohort 2, n=7) and 11 were evaluable for response (cohort 1, n=4; cohort 2, n=7). Median age was 61 years, 69.2% of patients were male, and median number of prior lines of treatment was 4 (range 2-8). The safety set included 8 DLBCL not otherwise specified (NOS), 1 high-grade B-cell lymphoma, 1 primary mediastinal B-cell lymphoma, and 3 patients with other subtypes, all T-cell rich. Cell of origin, determined locally, was reported in 10 patients (6 germinal center B-cell-like [GCB]; 4 non-GCB). At DCO, median duration of AZD4573 treatment was 4.0 weeks (range, 1-53) in cohort 1 and 12.6 weeks (range, 2-23) in cohort 2. AEs considered possibly related to either treatment occurred in 76.9% of patients and were grade ≥3 in 69.2% (Table 1). The most common AEs possibly related to AZD4573 were neutropenia (69.2%), thrombocytopenia (38.5%), alanine aminotransferase increased (38.5%) and aspartate aminotransferase increased (38.5%). Liver function test increases were short-lived with spontaneous resolution before subsequent weekly infusion. These findings were mainly due to down-modulation of hepatic transporter proteins and reduced clearance rather than hepatocellular impairment (based on clinical, in-vitro and in-silico assessments). Clinical tumor lysis syndrome (TLS) related to AZD4573 was not reported in cohort 1 and occurred in 1 of 7 patients in cohort 2 (TLS and transient 1.5xULN creatinine increase). No treatment-emergent AEs led to death. One patient in cohort 1 discontinued treatment due to grade 2 fatigue related to AZD4573. No dose limiting toxicities (DLTs) were observed in either cohort. The objective response rate (ORR) was 63.6% and the complete response (CR) rate was 36.4%. Of 4 response-evaluable patients in cohort 1, 1 had a CR and 1 had a partial response (PR), for an ORR of 50.0%. Of 7 evaluable patients in cohort 2, 3 had CRs and 2 had PRs, for an ORR of 71.4% (Table 2). Notably, 3/4 patients with prior CAR-T failure responded (2 CRs and 1 PR). Responses were seen in both GCB (1 CR/2 PRs in 5 evaluable patients) and non-GCB (2 CRs in 3 evaluable patients) subtypes. Conclusions: In this heavily pretreated population including CAR-T failures, the combination of AZD4573 and acalabrutinib resulted in high response rates (ORR 63.6%, CR rate 36.4%). No DLTs were detected, the combination was well tolerated with a manageable safety profile, and no new safety signals were identified. The study is currently enrolling in the Phase 2a expansion phase and updated data will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Structure-based rational design enables efficient discovery of a new selective and potent AKT PROTAC degrader

AKT and associated signaling pathways have been recognized as promising therapeutic targets for decades, and growing evidence indicates that inhibition or degradation of cellular AKT are viable strategies to treat cancer. Guided by an in silico modeling approach for rational linker design and based on our previous work in this field, we herein efficiently synthesized a small group of cereblon-recruiting AKT PROTAC molecules and identified a highly potent AKT degrader B4. Compared to the existing AKT degraders, B4 has a structurally unique AKT targeting warhead derived from the pyrazole-furan conjugated piperidine derivatives. It induces selective degradation of all three isoforms of AKT and exhibits efficacious anti-proliferation against several human hematological cancers. Notably, B4 demonstrates potent inhibition of AKT downstream signaling superior to its parental inhibitor. Together with its active analogs, B4 expands the arsenal of AKT chemical degraders as a valuable probe to uncover AKTs new functions and as a potential drug candidate to treat cancer.

Abstract P020: Schlafen 11 (SLFN11) as a predictive biomarker of the response to TAS1553, a novel small molecule ribonucleotide reductase subunit interaction inhibitor

Abstract Background: Ribonucleotide reductase (RNR) plays a crucial role in dNTP biosynthesis, which is required for DNA synthesis and repair, and is thought to be an attractive cancer therapeutic target. However, the precise significance of RNR inhibition remains to be elucidated, since reported RNR inhibitors exhibit limited pharmacological potency and off-target effects. We have developed a highly potent and novel small molecule RNR inhibitor, TAS1553, and reported that TAS1553 disrupted the protein-protein interaction between RNR subunits and exhibited the broad antiproliferative activity against human cancer cells in both in vitro and in vivo via oral administration. Phase-I study is currently ongoing and the identification of a predictive biomarker is essential to maximize clinical benefit of TAS1553. Here, we report a predictive biomarker of the response to TAS1553, and a clinical development strategy with the biomarker use. Material and methods: TAS1553 was synthesized at Taiho Pharmaceutical Co., Ltd. DNA replication stress, apoptosis, caspase activity and cellular growth inhibition induced by TAS1553 were assessed by western blotting, immunofluorescence staining, Caspase-Glo 3/7 assay and CellTiter-Glo® 2.0 assay, respectively. Cells were transfected with 2 nM siRNAs against SLFN11 (siSLFN11) and used for caspase-3/7 activation assay and cell proliferation assay. Results: TAS1553 induced intracellular pChk1, pRPA2 and γH2AX, followed by cleavage of PARP and caspase-3, suggesting that TAS1553 causes massive DNA replication stress. Then, we explored factors, involved in DNA replication stress, to predict response to TAS1553. Cytotoxicity profiling revealed broad antiproliferative activity of TAS1553 against both human hematological and solid cancer cell lines in a dose-​dependent manner (GI50 = 228-4150 nmol/L), and global gene expression profiling revealed that cells with high SLFN11 mRNA expression showed a higher sensitivity to TAS1553. TAS1553 exerted growth inhibitory activity without any cell killing effect against cells with low SLFN11 expression even at a concentration of 10 μmol/L, but cell killing activity against cells with high SLFN11 expression. Depletion of SLFN11 by siSLFN11 treatment in A673 cells which have high SLFN11 expression suppressed cytotoxic effect but not the growth inhibitory effect of TAS1553. Furthermore, we observed suppression of caspase-3/7 activation induced by TAS1553 in A673 cells transfected with siSLFN11. SLFN11 appeared to sensitize cancer cells to TAS1553 via promoting apoptosis. Conclusions: TAS1553, a novel orally available RNR inhibitor, showed potent antitumor activity in preclinical models of both hematological and solid tumors. Thus, TAS1553 could be a promising therapeutic agent for cancer, and SLFN11 could be a predictive biomarker in order to maximize clinical response to TAS1553. Citation Format: Hiroto Fukushima, Hiroyuki Ueno, Takuya Hoshino, Wakako Yano, Hiraku Itadani, Miki Terasaka, Sayaka Tsukioka, Takamasa Suzuki, Shoki Hara, Yoshio Ogino, Khoon Tee Chong, Tatsuya Suzuki, Yoshihiro Otsu, Satoshi Ito, Nozomu Tanaka, Seiji Miyahara. Schlafen 11 (SLFN11) as a predictive biomarker of the response to TAS1553, a novel small molecule ribonucleotide reductase subunit interaction inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P020.

A Phase I Study of GSK525762, a Selective Bromodomain (BRD) and Extra Terminal Protein (BET) Inhibitor: Results from Part 1 of Phase I/II Open Label Single Agent Study in Patients with Acute Myeloid Leukemia (AML)

A Phase I Study of GSK525762, a Selective Bromodomain (BRD) and Extra Terminal Protein (BET) Inhibitor: Results from Part 1 of Phase I/II Open Label Single Agent Study in Patients with Acute Myeloid Leukemia (AML)

Cancer-testis antigens in canine histiocytic sarcoma and other malignancies.

Cancer-testis antigens (CTAs) are a category of self proteins aberrantly expressed in diverse malignancies, mostly solid tumours, due to epigenetic de-repression. Normally expressed only in fetal or gametogenic tissues, CTAs are tantalizing immunotherapy targets, since autoimmunity risks appear minimal. Few prevalent CTAs have been identified in human hematologic cancers, and just two in their veterinary counterparts. We sought to discover new CTAs in canine hematologic cancers such as histiocytic sarcoma (HS) and lymphoma to foster immunotherapy development. To accomplish this, the ligandome binding the dog leukocyte antigen (DLA)-88*508:01 class I allele overexpressed in an HS line was searched by mass spectrometry to identify possible CTA-derived peptides, which could serve as CD8+ T-cell epitopes. Twenty-two peptides mapped to 5 human CTAs and 12 additional proteins with CTA characteristics. Expression of five promising candidates was then evaluated in tumour and normal tissue by quantitative and end-point RT-PCR. The ortholog of an established CTA, IGF2BP3, had unexpectedly high expression in peripheral blood mononuclear cells (PBMCs). Four other testis-enhanced proteins were also assessed. AKR1E2, SPECC1 and TPX2 were expressed variably in HS and T-cell lymphoma biopsies, but also at high levels in critical tissues, including kidney, brain and marrow, diminishing their utility. A more tissue-restricted candidate, NT5C1B, was detected in T-cell lymphomas, but also at low levels in some normal dog tissues. These results illustrate the feasibility of discovering canine CTAs by a reverse approach, proceeding from identification of MHC class I-presented peptides to a comparative RNA expression survey of tumours and normal tissues.

Pyronaridine exerts potent cytotoxicity on human breast and hematological cancer cells through induction of apoptosis.

The potent antimalarial drug pyronaridine (PND) was tested for its potential as an anticancer drug. After exposing cancerous (17) and non-cancerous (2) cells to PND for 72 hr, PND was found to exhibit consistent and potent cytotoxic activity at low micromolar (μM) concentrations that ranged from 1.6 μM to 9.4 μM. Moreover, PND exerted a significant selective cytotoxicity index (SCI) on five out of seven breast cancer cell lines tested, with favorable values of 2.5 to 4.4, as compared with the non-cancerous breast MCF-10A cell line. By using the same comparison, PND exhibited a significant SCI on three out of four leukemia/lymphoma cell lines with promising values of 3.3 to 3.5. One breast cancer and one leukemia cell line were tested further in order to determine the likely mode of action of PND. PND was found to consistently elicit phosphatidylserine externalization, mitochondrial depolarization, and DNA fragmentation, in both the triple negative MDA-MB-231 breast cancer and HL-60 leukemia cell lines. In addition, PND treatment altered cell cycle progression in both cancer cells. Subsequent DNA mobility-shift assays, UV-Visible spectroscopic titrations, and circular dichroism (CD) experiments revealed that PND intercalates with DNA. The findings presented in this study indicates that PND induces apoptosis and interfered with cell cycle progression of cancer cell lines and these results indicate that this drug has the potential as a repurposed drug for cancer therapy.

Targeted inhibition of human hematological cancers in vivo by doxorubicin encapsulated in smart lipoic acid-crosslinked hyaluronic acid nanoparticles

The chemotherapy of hematological cancers is challenged by its poor selectivity that leads to low therapeutic efficacy and pronounced adverse effects. Here, we report that doxorubicin encapsulated in lipoic acid-crosslinked hyaluronic acid nanoparticles (LACHA-DOX) mediate highly efficacious and targeted inhibition of human hematological cancers including LP-1 human multiple myeloma (MM) and AML-2 human acute myeloid leukemia xenografted in nude mice. LACHA-DOX had a size of ca. 183 nm and a DOX loading content of ca. 12.0 wt.%. MTT and flow cytometry assays showed that LACHA-DOX possessed a high targetability and antitumor activity toward CD44 receptor overexpressing LP-1 human MM cells and AML-2 human acute myeloid leukemia cells. The in vivo and ex vivo images revealed that LACHA-DOX achieved a significantly enhanced accumulation in LP-1 and AML-2 tumor xenografts. Notably, LACHA-DOX effectively suppressed LP-1 as well as AML-2 tumor growth and drastically increased mice survival rate as compared to control groups receiving free DOX or PBS. Histological analyses exhibited that LACHA-DOX caused little damage to the major organs like liver and heart. This study provides a proof-of-concept that lipoic acid-crosslinked hyaluronic acid nanoparticulate drugs may offer a more safe and effective treatment modality for CD44 positive hematological malignancies.

A Phase I/II Open-Label, Dose Escalation Study to Investigate the Safety, Pharmacokinetics, Pharmacodynamics and Clinical Activity of GSK525762 in Subjects with Relapsed, Refractory Hematologic Malignancies

Background: Bromodomains (BRDs) are domains found in a variety of proteins that recognize and bind to acetylated lysine residues in histone and other target proteins. The BRD and extra-terminal (BET) family of BRD-containing proteins bind to acetylated histone tails, alters chromatin structure and facilitates transcriptional complex localization to specific genes, thereby regulating gene transcription. The investigational agent GSK525762 is a potent small molecule inhibitor of the BET family of proteins that prevents assembly of macromolecular complexes and transcriptional response. GSK525762 inhibits growth in a broad spectrum of human hematological cancer cell lines, including cell lines derived from human patients with acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), and multiple myeloma (MM). GSK525762 is orally bio-available and can cause tumor reduction and improved animal survival in in vivo xenograft models of hematologic malignancies. Methods: This is a Phase I/II, open-label, 2-part study. Part 1 is a dose-escalation phase to determine the safety, tolerability, and recommended Phase 2 dose (RP2D) of GSK525762 in adult subjects with relapsed/refractory AML, NHL, and MM. Dose escalation is performed independently for each of these three cohorts. An accelerated dose titration was employed with one subject per dose level until the occurrence of a ≥Grade 2 drug-related toxicity or dose-limiting toxicity; thereafter, subjects have been enrolled in a standard 3+3 design. A Neuenschwander continual reassessment method (N-CRM) model is used at each dose escalation decision to provide guidance for the next dose escalation level. Starting dose is 5 mg GSK525762 orally once daily and dose escalation continues until the MTD is identified. All data, including safety, tolerability, pharmacokinetics (PK), and efficacy, are used to identify the RP2D. In Part 1, approximately 60-70 subjects will be enrolled (approximately 20 in each of three disease-specific cohorts); no hypothesis is being tested, and all analysis will be descriptive and exploratory. In Part 2, the clinical activity of GSK525762 (overall response rate) will be evaluated in expansion cohorts of subjects with AML, NHL, and MM. Up to 32 subjects may be enrolled into the AML and NHL cohorts, and up to 37 subjects may be enrolled in the MM cohort. Cohorts may be closed early if they do not exceed futility assessment. In addition, an exploratory cohort of subjects with double-hit lymphoma (DHL) and triple-hit lymphoma (THL) will be enrolled to evaluate clinical activity in this patient population. Additional study objectives include analysis of PK after single and repeat dosing, evaluation of pharmacodynamics (PD) and the relationship between GSK525762 exposure and safety/efficacy/PD parameters. Recruitment is ongoing across five centers (USA, UK, and Australia). Currently, 40 subjects have been enrolled (29 AML, 8 NHL, and 3 with MM).Study funded by GSK. DisclosuresStein:Seattle Genetics: Research Funding; Agios Pharmaceuticals: Other: Advisory Board, Research Funding; Celgene: Other: Advisory Board, Research Funding; Novartis: Consultancy. Huntly:Novartis: Speakers Bureau; BMS: Speakers Bureau; Ariad: Speakers Bureau; Pfizer: Speakers Bureau. Dickinson:GlaxoSmithKline: Consultancy, Research Funding. Horner:GlaxoSmithKline: Employment. Brennan:GlaxoSmithKline: Employment. Baron:GlaxoSmithKline: Employment. Kremer:GlaxoSmithKline: Employment, Equity Ownership. Dhar:GlaxoSmithKline: Employment.

Abstract IA14: Preclinical models for targeting oncogenic Ras signaling in cancer.

Abstract Somatic RAS mutations occur in 30% of human cancers, and are common in both melanoma and hematopoietic cancers. Importantly, whereas KRAS mutations predominate in epithelial cancers, NRAS is mutated much more often in melanoma and hematopoietic cancers. The most obvious therapeutic strategy for cancers with RAS mutations – developing specific inhibitors of the mutant protein – is extremely challenging due to the very high affinity of Ras for GTP, structural considerations of the Ras/GTPase activating protein (Ras/GAP) molecular switch, and the loss of normal enzymatic activity as a consequence of oncogenic RAS mutations. Given these issues, most recent drug discovery efforts have focused on inhibiting downstream biochemical targets of mutant Ras such as Raf, MEK, and Akt with many compounds undergoing clinical evaluation. We have exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human hematologic cancers and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable myeloid and lymphoid leukemias characterized by hyperactive Ras signaling. We utilize cohorts of mice that are engrafted with the same primary leukemias for preclinical testing. In this presentation, I will focus on three biologic and therapeutic questions related to NRAS in tumorigenesis: (1) loss of the normal NRAS allele in cancers with oncogenic NRAS mutations; (2) responses of primary Nras mutant leukemias to MEK and PI3 kinase inhibitors; and, (3) potential strategies for selectively targeting oncogenic N-Ras in cancer. Note: This abstract was presented as part of the Joint Session: Clinical Science Interactions between Melanoma and Hematologic Malignancies with the AACR Special Conference on Advances in Melanoma: From Biology to Therapy held September 20-23, 2014 in Philadelphia, PA. Citation Format: Kevin Shannon. Preclinical models for targeting oncogenic Ras signaling in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA14.

Abstract 5397: Characterization of INCB053914, a novel pan-PIM kinase inhibitor

Abstract The PIM (Proviral Integration sites for Moloney virus) family of constitutively active serine/threonine kinases consists of three members (PIM1, PIM2 and PIM3) that phosphorylate substrates involved in both apoptosis and cellular metabolism. As downstream targets of the JAK/STAT pathway and by having overlapping substrates with the PI3K/AKT pathway, PIM kinases integrate signals from multiple pathways important for the survival and proliferation of malignant cells. Overexpression of PIM kinases has been reported in human hematological cancers with each isoform showing a distinct expression pattern among the various malignancy subtypes, including MM, AML, ALL and NHL. Although the three family members possess overlapping functions that are mutually compensatory, mice deficient for all three PIM kinases exhibit minor phenotypic abnormalities, with smaller overall size at maturity and decreased cellular responses to cytokines. These data suggest that pan-inhibition of the PIM kinase family will have a favorable toxicity profile and may be required for therapeutic benefit in hematological malignancies. INCB053914 is a novel, ATP-competitive, small molecule, pan-inhibitor of PIM kinases. In biochemical assays, INCB053914 is potent against PIM1, PIM2 and PIM3 and highly selective. In cell proliferation assays, INCB053914 is active as a single agent in the majority of cell lines derived from different hematological malignancies, including MM, AML, DLBCL, MCL and T-ALL, with IC50 values ranging from 3 - 300 nM. INCB053914 synergizes with a variety of cytotoxic and targeted agents, reducing the viability of a panel of hematological tumor cell lines. In pharmacodynamic assays, INCB053914 inhibits phosphorylation of S6RP, P70S6K, 4E-BP-1 and BAD, known PIM kinase targets. Following treatment with INCB053914, induction of PIM kinase protein levels was noted in human cancer cell lines. Importantly, this compensatory response does not reduce the activity of the molecule, as effects on downstream signaling and efficacy remain intact when PIM kinase levels are elevated. Induction of PIM kinases after INCB53914 treatment was also evident in whole blood from healthy volunteers and in primary blast cells isolated from patients diagnosed with AML, further supporting the need for pan-PIM kinase inhibition. Taken together, these results indicate that INCB053914 has potent and selective activity against PIM kinases and may be useful as monotherapy or in combination with other agents for treating hematologic malignancies that are dependent on PIM kinase activity for growth and survival. Citation Format: Niu Shin, Maryanne Covington, Richard Wynn, Yanlong Li, Alexander Margulis, Qian Wang, Kathy Wang, Cindy Marando, Patricia Feldman, Lynn Leffet, Karen Gallagher, Xin He, Hong Chang, He Zhang, Hao Feng, Yun-Long Li, Chu-Biao Xue, Wenqing Yao, Timothy Burn, Kris Vaddi, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Gregory Hollis, Robert Newton, Reid Huber, Peggy Scherle, Holly Koblish. Characterization of INCB053914, a novel pan-PIM kinase inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5397. doi:10.1158/1538-7445.AM2015-5397

Abstract IA12: Preclinical models for targeting oncogenic Ras signaling in cancer

Abstract Somatic RAS mutations occur in 30% of human cancers, and are common in both melanoma and hematopoietic cancers. Importantly, whereas KRAS mutations predominate in epithelial cancers, NRAS is mutated much more often in melanoma and hematopoietic cancers. The most obvious therapeutic strategy for cancers with RAS mutations – developing specific inhibitors of the mutant protein – is extremely challenging due to the very high affinity of Ras for GTP, structural considerations of the Ras/GTPase activating protein (Ras/GAP) molecular switch, and the loss of normal enzymatic activity as a consequence of oncogenic RAS mutations. Given these issues, most recent drug discovery efforts have focused on inhibiting downstream biochemical targets of mutant Ras such as Raf, MEK, and Akt with many compounds undergoing clinical evaluation. We have exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human hematologic cancers and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable myeloid and lymphoid leukemias characterized by hyperactive Ras signaling. We utilize cohorts of mice that are engrafted with the same primary leukemias for preclinical testing. In this presentation, I will focus on three biologic and therapeutic questions related to NRAS in tumorigenesis: (1) loss of the normal NRAS allele in cancers with oncogenic NRAS mutations; (2) responses of primary Nras mutant leukemias to MEK and PI3 kinase inhibitors; and, (3) potential strategies for selectively targeting oncogenic N-Ras in cancer. Note: This abstract was presented as part of the Joint Session: Clinical Science Interactions between Melanoma and Hematologic Malignancies with the AACR Special Conference: Hematologic Malignancies: Translating Discoveries to Novel Therapies held September 20-23, 2014 in Philadelphia, PA. Citation Format: Kevin Shannon. Preclinical models for targeting oncogenic Ras signaling in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA12.

Synthesis and antiproliferative activity of α-branched α,β-unsaturated ketones in human hematological and solid cancer cell lines

A series of α-branched α,β-unsaturated ketones were prepared via boron trifluoride etherate mediated reaction between arylalkynes and carboxaldehydes. The evaluation of the antiproliferative activity over hematological (NB4) and solid cancer (A549, MCF-7) cell lines provided a structure-activity relationship. 5-Parameter QSAR equations were built which were able to explain 80%–92% of the variance in activity. The resulting selective lead compound showed IC50 value 0.6 μM against the hematological cell line and did not cause apoptosis, but blocked cell cycle in G0/G1. Moreover, it was demonstrated that this compound enhances and accelerates retinoic acid induced granulocytic differentiation.

Novel Role for NFAT3 in ERK-Mediated Regulation of CXCR4

The G-protein coupled chemokine (C-X-C motif) receptor CXCR4 is linked to cancer, HIV, and WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. While CXCR4 is reported to be overexpressed in multiple human cancer types and many hematological cancer cell lines, we have observed poor in vitro cell surface expression of CXCR4 in many solid tumor cell lines. We explore further the possible factors and pathways involved in regulating CXCR4 expression. Here, we showed that MEK-ERK signaling pathway and NFAT3 transcriptional factor plays a novel role in regulating CXCR4 expression. When cultured as 3D spheroids, HeyA8 ovarian tumor cells showed a dramatic increase in surface CXCR4 protein levels as well as mRNA transcripts. Furthermore, HeyA8 3D spheroids showed a decrease in phospho-ERK levels when compared to adherent cells. The treatment of adherent HeyA8 cells with an inhibitor of the MEK-ERK pathway, U0126, resulted in a significant increase in surface CXCR4 expression. Additional investigation using the PCR array assay comparing adherent to 3D spheroid showed a wide range of transcription factors being up-regulated, most notably a> 20 fold increase in NFAT3 transcription factor mRNA. Finally, chromatin immunoprecipitation (ChIP) analysis showed that direct binding of NFAT3 on the CXCR4 promoter corresponds to increased CXCR4 expression in HeyA8 ovarian cell line. Taken together, our results suggest that high phospho-ERK levels and NFAT3 expression plays a novel role in regulating CXCR4 expression.

Abstract 4531: INCB40093 is a selective PI3Kδ inhibitor with potent antiproliferative activity against human B-cell tumors

Abstract Phosphatidylinositol 3-kinases (PI3Ks) belong to a family of lipid signaling kinases that phosphorylate phosphatidylinositol-4,5-bisphosphate, giving rise to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and functions by mediating the signaling of key receptors on B cells including BCR, BAFF-R, IL-4R, CD40 and TLRs. In addition, increased malignant B cell proliferation and survival has been associated with aberrant activation of PI3Kδ, and data from recent clinical studies using idelalisib, a PI3Kδ specific inhibitor, indicate that the enzyme is a therapeutic target for the treatment of B cell lymphomas. INCB40093 is a novel, potent small molecule inhibitor of PI3Kδ with 74 to > 900-fold selectivity over the other PI3K family members. It also does not inhibit (< 30% inhibition) a broad panel of 196 other kinases when tested at a concentration of 1 μM. INCB40093 blocks the proliferation of primary B cells induced by the activation of PI3Kδ-mediated signaling through several key receptors (e.g. BCR, CD40). More importantly, INCB40093 is potent (IC50 values of ∼10-100 nM) in cell-based assays relevant to the pathogenesis of B cell malignancies, such as PI3Kδ-mediated signaling and growth of human B cell lines. This effect is not due to general cytotoxicity because 10 μM INCB40093 had no significant effect on the growth of non-lymphoid cell lines. Compared to the inhibition of primary B cell proliferation, INCB40093 is at least 50 times less potent in assays that measure human T cell or NK cell proliferation, and neutrophil and macrophage function, suggesting that the impact of INCB40093 on the human immune system will likely be restricted to B cells. In vivo, the effects of INCB40093 were evaluated in two human hematological cancer xenograft models. In the Pfeiffer model of non-Hodgkin's lymphoma, oral administration of INCB40093 inhibited tumor growth as a single agent. INCB40093 also potentiated the anti-tumor effects of the clinically relevant chemotherapeutic agent, bendamustine. The tumor growth inhibitory effects of INCB40093 were also demonstrated in a myeloma tumor xenograft model, INA-6.Tu1. Taken together, pharmacological data obtained in both in vitro and in vivo model systems support the potential utility of orally administered INCB40093 in the treatment of B cell malignancies. Citation Format: Niu Shin, Kathy Wang, Leslie Hall, Qian Wang, Gengjie Yang, Yanlong Li, Yun-Long Li, Maryanne Covington, Jordan Fridman, Robert Newton, Peggy Scherle. INCB40093 is a selective PI3Kδ inhibitor with potent antiproliferative activity against human B-cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4531. doi:10.1158/1538-7445.AM2014-4531

From the covalent linkage of drugs to novel inhibitors of ribonucleotide reductase: Synthesis and biological evaluation of valproic esters of 3′-C-methyladenosine

We synthesized a series of serum-stable covalently linked drugs derived from 3′-C-methyladenosine (3′-Me-Ado) and valproic acid (VPA), which are ribonucleotide reductase (RR) and histone deacetylase (HDAC) inhibitors, respectively. While the combination of free VPA and 3′-Me-Ado resulted in a clear synergistic apoptotic effect, the conjugates had lost their HDAC inhibitory effect as well as the corresponding apoptotic activity. Two of the analogs, 2′,5′-bis-O-valproyl-3′-C-methyladenosine (A160) and 5′-O-valproyl-3′-C-methyladenosine (A167), showed promising cytotoxic activities against human hematological and solid cancer cell lines. A167 was less potent than A160 but had interesting features as an RR inhibitor. It inhibited RR activity by competing with ATP as an allosteric effector and concomitantly reduced the intracellular deoxyribonucleoside triphosphate (dNTP) pools. A167 represents a novel lead compound, which in contrast to previously used RR nucleoside analogs does not require intracellular kinases for its activity and therefore holds promise against drug resistant tumors with downregulated nucleoside kinases.