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We present a design study into an ion trap electrode geometry for applying near-field microwave two-qubit gates. This design features an ‘S’-shaped meander electrode to passively null the microwave field. It has ground planes separating the meander electrode from all of the DC and single-qubit microwave electrodes, which should reduce the sensitivity of the microwave field distribution to the boundary conditions of these electrodes. We show that it is possible to design a single-layer trap with this geometry such that the simulated microwave field null overlaps with the RF field null, and that the positions of these nulls can be simulated to a precision of 100 nm with moderate computing resources. We also show that such a trap can be designed such that ion chains can be trapped, transported and split with feasible DC and RF voltages. While this particular design is optimized for $$^{43}$$ 43 Ca$$^{+}$$ + ions, our approach could be applied to other ions by changing the microwave frequency to match the corresponding qubit transition frequency.

Supplementary Figures S1-S7 with captions and Supplementary Tables

Supplementary Methods

PDF file - 82K, Supplementary Table 1: Fold changes of NF-κB target genes in CLL: CLL cells (n=12) were treated with 300 nM CFZ (1 hr), 1.7 �M CpG (4 hr) or 500 ng/ml CD40L (4 hr). RNA was extracted at 8 hr from CFZ treated samples and mRNA expression was analyzed using real-time RT-PCR. Cells highlighted in light grey have estimated fold changes > 2 and were significant at the (unadjusted) α=0.05 level of significance. Cells highlighted in dark grey are significant at the α=0.05 level, but have estimated fold changes < 2. Supplementary Figure 1: Selection of 697 cells to generate p53DN cells: Jeko, MEC-1 and 697 cells (n=3) were exposed to 0, 300 nM CFZ for 1 hr and 5 Gy gamma irradiation. (S1A) Viability was assessed by MTS assay at 48 hrs and results were calculated relative to time-matched samples untreated samples. (S1B) Whole cell lysates and RNA were collected at 8 hr and were immunoblotted for indicated proteins using GAPDH as a loading control. (S1C) Changes in transcription of p53 and subsequent targets were assessed by real-time RT-PCR.

Purpose: The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2, and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells.Experimental Design: Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction, and electrophoretic mobility shift assays. In addition, a p53 dominant-negative construct was generated in a human B-cell line.Results: Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells versus CLL. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IκBα, phosphorylation of IκBα, and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription.Conclusions: Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support phase I investigation of CFZ in this disease. Clin Cancer Res; 19(9); 2406–19. ©2013 AACR.

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Diffuse large B-cell lymphoma (DLBCL) is the most common non- Hodgkin lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front-line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin b-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signaling pathway by binding to β-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, β-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

Hairy cell leukemia (HCL) is a rare B-cell malignancy, and there is a need for novel treatments for patients who do not benefit from purine analogs. Ibrutinib, an oral agent targeting Bruton tyrosine kinase in the B-cell receptor signaling pathway, is highly effective in several malignancies. Its activity in HCL was unknown, so we conducted a multisite phase 2 study of oral ibrutinib in patients with either relapsed classic or variant hairy cell leukemia. The primary outcome measure was the overall response rate (ORR) at 32 weeks, and we also assessed response at 48 weeks and best response during treatment. Key secondary objectives were characterization of toxicity and determination of progression-free survival (PFS) and overall survival (OS). Thirty-seven patients were enrolled at 2 different doses (24 at 420 mg, 13 at 840 mg). The median duration of follow-up was 3.5 years (range, 0-5.9 years). The ORR at 32 weeks was 24%, which increased to 36% at 48 weeks. The best ORR was 54%. The estimated 36-month PFS was 73% and OS was 85%. The most frequent adverse events were diarrhea (59%), fatigue (54%), myalgia (54%), and nausea (51%). Hematologic adverse events were common: anemia (43%), thrombocytopenia (41%), and neutropenia (35%). Ibrutinib can be safely administered to patients with HCL with objective responses and results in prolonged disease control. Although the initial primary outcome objective of the study was not met, the observation of objective responses in heavily pretreated patients coupled with a favorable PFS suggests that ibrutinib may be beneficial in these patients. This trial was registered at www.clinicaltrials.gov as #NCT01841723.

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123- lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a "bridge-to-transplant" before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.

Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or de novo expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel in vitro and in vivo pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in preclinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of BRD4 profiling in other cancers. Significance: To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. Cancer Discov; 8(4); 458–77. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 371

Epstein-Barr Virus (EBV) is an oncogenic herpes virus associated with the development of malignancies associated with poor outcomes. Current treatments lead to further immune suppression, increasing the risk of EBV reactivation and other opportunistic infections. Strategies aimed at reversing the immune-suppressive microenvironment are likely to result in more durable responses while avoiding deleterious effects of chemotherapy. It is well-established that macrophages play an important role in inhibiting the anti-tumor response as evidenced by poor prognosis associated with increasing tumor-associated macrophage (TAM) density or lower ratio of cytotoxic T lymphocytes. Here we describe a pre-clinical model showing the expansion of cytotoxic TAMs and potential mechanisms to prevent this immunosuppressive environment. To assess soluble factors produced by the interaction of tumor on immune effector cells, we used a co-culture (CoCx) system of EBV-transformed human B cell lymphoblastoid cell lines (LCL) cultured with autologous peripheral blood mononuclear cells (PBMC). Serum-free medium collected after 48 hrs showed increased myeloid-associated factors which were absent from either the LCL or PBMC cultures, suggesting the interaction of tumor cells with immune effectors was leading to the production of chemokines known to attract myeloid cells. When purified autologous CD14+ monocytes were incubated in the presence of CoCx conditioned medium (CM) they generated proliferative foci that were absent in the LCL or PBMC CM alone. To better understand which cellular subsets were necessary for the release of these soluble factors, immune subsets were removed from PBMC prior to incubation with LCL or purified and incubated with LCL. We found that T cells, (CD8+ and CD4+), when incubated in the presence of LCL were necessary and sufficient to stimulate the proliferation of myeloid cells. CD56+, CD14+, or CD19+ subsets within the CM did not appear to contribute to the proliferation of purified myeloid cells. We next used flow cytometry to characterize the monocyte-derived population that expanded when introduced to CoCx CM. We again isolated autologous CD14+ monocytes from PMBC and incubated them for 6 days with CoCx CM followed by immunophenotyping. The monocyte-derived population retained CD14 and CD11b in addition to showing pronounced increases in both canonical M1 and M2 macrophage markers (HLA-DR, and CD163 respectively), and PD-L1, indicating activation consistent with a TAM phenotype. To characterize the functional consequence of this cell population, CoCx CM-derived macrophages were incubated with autologous T cells that were either activated non-specifically with anti-CD3/C28 or left inactivated. Surprisingly, we noted a marked decrease in viable activated T cells (both CD4+ and CD8+) when exposed to conditioned macrophages. However, the inactivated T cell populations were only mildly effected. Together, with previously described experiments, this suggests a mechanism by which exposure of T cells to EBV+ lymphoblastic tumor lines results in cytokine release, polarizing monocytes to TAMs that, in turn, exhibit cytotoxic activity to tumor-specific T cells. The precise mechanism of cytotoxicity is under investigation. We previously reported the eIF4A translational inhibitor silvestrol potentiated a potent immune-mediated anti-tumor response against EBV-driven lymphoma mainly by promoting expansion and activity of EBV-specific cytotoxic T cells. We next questioned if macrophages were important to this mechanism. We found that with silvestrol, when added to CoCx, the resultant CM did not drive the proliferation of TAMs, and Immunophenotype of CoCx showed silvestrol treatment to result in the ablation of CD14+ cells from culture. Interrogation by transcriptome analysis indicated a potential role of aryl hydrocarbon receptor (AhR). Protein analysis by western blotting indicated an increase of AhR in isolated monocytes when exposed to CoCx CM, which was abrogated by addition of silvestrol. Together these results suggest AhR may play a role in the polarization of macrophages within the tumor microenvironment leading to elimination of tumor-specific T cells. While full mechanistic characterization is ongoing, these preliminary results provide a potentially novel approach to modulate tumor specific immunity in this challenging group of EBV-related malignancies. Disclosures Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Mutated mitogen-activated protein kinase (MAPK) pathway components promote tumor survival, proliferation, and immune evasion in solid tumors. MAPK mutations occur in hematologic cancers as well, but their role is less clear and few models are available to study this. We developed an in vivo model of disseminated BRAFV600E B-cell leukemia to determine the effects of this mutation on tumor development and immune evasion. Mice with B-cell-restricted BRAFV600E expression crossed with the Eµ-TCL1 model of chronic lymphocytic leukemia (CLL) developed leukemia significantly earlier (median, 4.9 vs 8.1 months; P < .001) and had significantly shorter lifespan (median, 7.3 vs 12.1 months; P < .001) versus BRAF wild-type counterparts. BRAFV600E expression did not affect B-cell proliferation but reduced spontaneous apoptosis. BRAFV600E-mutant leukemia produced greater T-cell effects, evidenced by exhaustion immunophenotype and CD44+ T-cell percentage, as well as increased expression of PD-L1 on CD11b+ cells. Results were confirmed in syngeneic mice engrafted with BRAFV600E leukemia cells. Furthermore, a BRAFV600E-expressing CLL cell line more strongly inhibited anti-CD3/CD28-induced T-cell proliferation, which was reversed by BRAFV600E inhibition. These results demonstrate the immune-suppressive impact of BRAFV600E in B-cell leukemias and introduce a new model to develop rational combination strategies targeting both tumor cells and tumor-mediated immune evasion.

Variant hairy cell leukemia (vHCL) is an uncommon disorder accounting for 10% of all HCL cases [1]. The difference in therapeutic response to chemotherapy between patients with classic HCL (cHCL) a...

To the editor: Chronic lymphocytic leukemia (CLL) exhibits an indolent precursor phase prior to development of a more aggressive phenotype which requires treatment. Understanding the early pathogenesis of CLL offers the opportunity to better implement more effective intervention for this disease.

Somatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53-/- mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53-/- bone marrow cells rapidly develop a highly penetrant AML. We find that p53-/- cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53-/- MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53-/- synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.

Three new (1-3) and two known (4 and 5) cytotoxic cardiac glycosides were isolated and characterized from a medicinal plant, Streblus asper Lour. (Moraceae), collected in Vietnam, with six new analogues and one known derivative (5a-g) synthesized from (+)-strebloside (5). A preliminary structure-activity relationship study indicated that the C-10 formyl and C-5 and C-14 hydroxy groups and C-3 sugar unit play important roles in the mediation of the cytotoxicity of (+)-strebloside (5) against HT-29 human colon cancer cells. When evaluated in NCr nu/nu mice implanted intraperitoneally with hollow fibers facilitated with either MDA-MB-231 human breast or OVCAR3 human ovarian cancer cells, (+)-strebloside (5) showed significant cell growth inhibitory activity in both cases, in the dose range 5-30 mg/kg.

Epigenetic or transcriptional silencing of important tumor suppressors has been described to contribute to cell survival and tumorigenesis in chronic lymphocytic leukemia (CLL). Using gene expression microarray analysis, we found that thousands of genes are repressed more than 2-fold in CLL compared to normal B cells; however therapeutic approaches to reverse this have been limited in CLL. Following treatment with the Hsp90 inhibitor 17-DMAG, a significant number of these repressed genes were significantly re-expressed. One of the genes significantly repressed in CLL and up-regulated by 17-DMAG was suppressor of cytokine signaling 3, (SOCS3). SOCS3 has been shown to be silenced in solid tumors as well as myeloid leukemia; however little is known about the regulation in CLL. We found that 17-DMAG induces expression of SOCS3 by via the activation of p38 signaling, and subsequently inhibits AKT and STAT3 phosphorylation resulting in downstream effects on cell migration and survival. We therefore suggest that SOCS3 is an important signaling protein in CLL, and Hsp90 inhibitors represent a novel approach to target transcriptional repression in B cell lymphoproliferative disorders which exhibit a substantial degree of gene repression.

Fludarabine (F) and cyclophosphamide (C) remain backbones of up-front chemotherapy regimens for chronic lymphocytic leukemia (CLL). We report long-term follow-up of a randomized F versus FC trial in untreated, symptomatic CLL (NCT00003764, clinicaltrials.gov). With median follow-up of 88 months, estimated median progression-free survival (PFS) was 19.3 versus 48.1 months for F (N=109) and FC (N=118) respectively (p

The activating BRAF mutation p.V600E has been identified in many human cancers, including colon and lung adenocarcinoma, papillary thyroid cancer, malignant melanoma, and hairy cell leukemia. The latter two are of particular interest both because of the high proportion of cases harboring the mutation as well as the dramatic responses to BRAF inhibitor therapy which have been reported in the literature. Here we report for the first time the case of a patient with hairy cell leukemia and malignant melanoma both harboring the BRAF V600E mutation, and the successful treatment of both cancers with the BRAF inhibitor dabrafenib.

Mutated mitogen-activated protein kinase (MAPK) pathway components promote tumor survival, proliferation, and immune evasion in solid tumors. MAPK mutations occur in hematologic cancers as well, but their role is less clear and few models are available to study this. We developed an in vivo model of disseminated BRAF

Long non-coding ribonucleic acids (RNAs) are a novel class of RNA molecules, which are increasingly recognized as important molecular players in solid and hematologic malignancies. Herein we investigated whether long non-coding RNA expression is associated with clinical and molecular features, as well as outcome of younger adults (aged60 years) with

Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.

As part of an ongoing project to explore filamentous fungi for anticancer and antibiotic leads, eleven compounds were isolated and identified from an organic extract of the fungus Scytalidium album (MSX51631) using bioactivity-directed fractionation against human cancer cell lines. Of these, eight were a series of sorbicillinoid analogues (1–8), of which four were new [scalbucillin A (2), scalbucillin B (3), scalbucillin C (6), and scalbucillin D (8)], two were phthalides (9–10), and one was naphthalenone (11). Compounds (1–11) were tested in the MDA-MB-435 (melanoma) and SW-620 (colon) cancer cell lines. Compound 1 was the most potent with IC50 values of 1.5 and 0.5 μM, respectively, followed by compound 5, with IC50 values of 2.3 and 2.5 μM at 72 h. Compound 1 showed a 48-h IC50 value of 3.1 μM when tested against the lymphocytic leukemia cell line OSU-CLL, while the nearly identical compound 5 had almost no activity in this assay. Compounds 1 and 5 showed selective and equipotent activity against Aspergillus niger with minimum inhibitory concentration values of 0.05 and 0.04 μg/ml (0.20 and 0.16 μM), respectively. The in vitro hemolytic activity against sheep erythrocytes of compounds 1 and 5 was investigated and were found to provoke 10% hemolysis at 52.5 and 45.0 μg/ml, respectively, indicative of a promising safety factor.

Treatment options for patients with Epstein-Barr Virus-driven lymphoproliferative diseases (EBV-LPD) are limited. Chemo-immunotherapeutic approaches often lead to immune suppression, risk of lethal infection and EBV reactivation, thus it is essential to identify agents that can deliver direct anti-tumor activity while preserving innate and adaptive host immune surveillance. Silvestrol possesses direct anti-tumor activity in multiple hematologic malignancies while causing minimal toxicity to normal mononuclear cells. However, the effects of silvestrol on immune function have not been described. We utilized in vitro and in vivo models of EBV-LPD to simultaneously examine the impact of silvestrol on both tumor and normal immune function. We show that silvestrol induces direct anti-tumor activity against EBV-transformed lymphoblastoid cell lines (LCL), with growth inhibition, decreased expression of the EBV oncogene latent membrane protein-1, and inhibition of the downstream AKT, STAT1 and STAT3 signaling pathways. Silvestrol promoted potent indirect anti-tumor effects by preserving expansion of innate and EBV antigen-specific adaptive immune effector subsets capable of effective clearance of LCL tumor targets in autologous co-cultures. In an animal model of spontaneous EBV-LPD, silvestrol demonstrated significant therapeutic activity dependent on the presence of CD8-positive T-cells. These findings establish a novel immune-sparing activity of silvestrol, justifying further exploration in patients with EBV-positive malignancies.

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

Recent progress is described in an ongoing collaborative multidisciplinary research project directed towards the purification, structural characterization, chemical modification, and biological evaluation of new potential natural product anticancer agents obtained from a diverse group of organisms, comprising tropical plants, aquatic and terrestrial cyanobacteria, and filamentous fungi. Information is provided on how these organisms are collected and processed. The types of bioassays are indicated in which initial extracts, chromatographic fractions, and purified isolated compounds of these acquisitions are tested. Several promising biologically active lead compounds from each major organism major class investigated are described, and these may be seen to be representative of very wide chemical diversity.

Background: Hairy Cell Leukemia (HCL) is a rare hematological malignancy, comprising only of 2% of all leukemias, with an estimated 900 new cases diagnosed each year in the United States. HCL displays a characteristic immunophenotypic profile that include pan-B cell markers including CD103, CD11c, and CD25. World Health Organization guidelines defines two forms of HCL, classic HCL (cHCL) and variant HCL (HCLv) as two distinct clinical entities. Patients with cHCL have a distinct immunophenotypic profile on their malignant leukemic cells including CD20+, CD19+, CD11c+, CD25+, CD103+, and CD123+, while the leukemic cells from patients with HCLv show CD11c+, CD20+ and CD19+, while lacking CD25 and CD123 expression. Some patients with cHCL will retain CD25 positivity while demonstrating negativity for other typical markers, herein termed atypical HCL (aHCL). Presence or absence of CD25 is an important determinant in classifying patients into cHCL and HCLv. Although it has previously been reported that CD25 expression may be lost during treatment with the targeted agent vemurafenib, we sought to identify whether this immunophenotypic change occurs following other treatment types, including standard purine nucleoside analog therapy and with targeted BTK inhibition. Methods: Adult patients (≥18 years) with a diagnosis of HCL whom had immunophenotype data collected before and after treatment between 2010 and 2018 were included in the study. Immunophenotype and morphological characteristics of initial and follow-up peripheral blood, bone marrow aspirate, and core biopsy specimens were reviewed and correlated with the treatment received. Results: We evaluated 30 HCL patients who underwent different therapies. All available specimens were reviewed and showed morphologic features characteristic for cHCL (n=26, 86.7%), and aHCL (n=4, 13.3%). The median age at HCL diagnosis was 50 years (44-76 years) with male predominance (76%). Patients with aHCL were treated with ibrutinib (n=2) and pentostatin (n=2). Patients with cHCL were treated with pentostatin (n=12), ibrutinib (n=8), vemurafenib (n=4), dabrafenib (n=1), and cladribine (n=1). Bone marrow analyses showed that all the patients had leukemic B-lymphocyte co-expression of CD19, CD20, CD103, CD11c, CD25, and CD123 prior to treatment. Some patients also had a smaller percentage of lymphocytes lacking CD25 expression along with the CD25 positive lymphocytes. Follow-up bone marrow and peripheral blood analysis showed that almost half (n=14, 46%) of treated patients had a partial or complete loss of CD25 expression regardless of the treatment type. Leukemic cells continued to express other HCL signature markers. Conclusion: Our study indicates that during the course of disease some patients display a loss of CD25 expression after therapy. This phenomenon was observed across different therapies and is not specific to the type of treatment. This is the first study to show treatment-dependent CD25 variability with pentostatin, ibrutinib and dabrafenib. Our results advocate for caution when using CD25 for the differential diagnosis of cHCL versus HCLv in treated patients. Future studies are needed in larger patient cohorts to determine the overall role and utility of CD25 in the diagnosis of cHCL and HCLv. Disclosures Lozanski: Genentech: Research Funding; Stem Line: Research Funding; BI: Research Funding; Novartis: Research Funding; Beckman: Research Funding; Coulter: Research Funding. Andritsos:Astra Zeneca: Consultancy; HCLF: Membership on an entity's Board of Directors or advisory committees.

Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5′ untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.

Four new flavanones, designated as (+)-5″-deacetylpurpurin (1), (+)-5-methoxypurpurin (2), (2S)-2,3-dihydrotephroglabrin (3), and (2S)-2,3-dihydrotephroapollin C (4), together with two known flavanones (5 and 6), three known rotenoids (7-9), and one known chalcone (10) were isolated from a chloroform-soluble partition of a methanol extract from the combined flowers, fruits, leaves, and twigs of Indigofera spicata, collected in Vietnam. The compounds were obtained by bioactivity-guided isolation using the HT-29 human colon cancer, 697 human acute lymphoblastic leukemia, and Raji human Burkitt's lymphoma cell lines. The structures of 1-4 were established by extensive 1D- and 2D-NMR experiments, and the absolute configurations were determined by the measurement of specific rotations and CD spectra. The cytotoxic activities of the isolated compounds were tested against the HT-29, 697, Raji, and CCD-112CoN human normal colon cells. Also, the quinone reductase induction activities of the isolates were determined using the Hepa 1c1c7 murine hepatoma cell line. In addition, cis-(6aβ,12aβ)-hydroxyrotenone (7) was evaluated in an in vivo hollow fiber bioassay using HT-29, MCF-7 human breast cancer, and MDA-MB-435 human melanoma cells.

Purpose: The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2, and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells. Experimental Design: Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction, and electrophoretic mobility shift assays. In addition, a p53 dominant-negative construct was generated in a human B-cell line. Results: Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells versus CLL. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IκBα, phosphorylation of IκBα, and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription. Conclusions: Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support phase I investigation of CFZ in this disease. Clin Cancer Res; 19(9); 2406–19. ©2013 AACR.

The potentiating action of the flavonolignan, (-)-hydnocarpin, in combination with vincristine was evaluated in the 697 acute lymphoblastic leukemia cell line and a P-gp-expressing variant, 697-R. Vincristine at 3 nM caused nearly complete growth inhibition in 697 cells, versus a 17% growth inhibition in 697-R cells. When combined with (-)-hydnocarpin at concentrations of 10 and 5 µM, vincristine-mediated growth inhibition in the 697-R cells increased significantly over the sum of the individual agents to 72% (p ≤ 0.0001), and 41% (p = 0.0256), respectively. Vincristine at 1.5 nM (66% growth inhibition) and 0.75 nM (39% growth inhibition) combined with (-)-hydnocarpin at 10 µM (42% growth inhibition) in the 697 cells, caused a significant increase in growth inhibition to 83% (p = 0.03) and to 61% (p < 0.0001), respectively, when compared to vincristine treatment as a single agent. To investigate the mechanism for the vincristine re-sensitization caused by (-)-hydnocarpin, the P-gp inhibitory effect of (-)-hydnocarpin was evaluated.

Somatic mutation of the DNMT3A gene at the arginine R882 site is common in acute myeloid leukaemia (AML). The prognostic significance of DNMT3A R882 mutation clearance, using traditional diagnostic next generation sequencing (NGS) methods, during complete remission (CR) in AML patients is controversial. We examined the impact of clearing DNMT3A R882 mutations at diagnosis to the detectable threshold of ˂3% during CR on outcome in 56 adult AML patients. Mutational remission, defined as clearance of pre-treatment DNMT3A R882 and all other AML-associated mutations to a variant allele frequency ˂3%, occurred in 14 patients whereas persistent DNMT3A R882 mutations were observed in 42 patients. There were no significant differences in disease-free or overall survival between patients with and without DNMT3A R882 mutation clearance. Patients with persistent DNMT3A R882 who cleared all other AML mutations and did not acquire new mutations (n = 30), trended towards longer disease-free survival (1·6 vs. 0·6 years, P = 0·06) than patients with persistence of DNMT3A R882, in addition to other mutations or acquisition of new AML-associated mutations, such as those in TET2, JAK2, ASXL1 and TP53 (n = 12). These data demonstrate that DNMT3A R882 mutations, as assessed by traditional NGS methods, persist in the majority of AML patients in CR.

Purpose: During cell-cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of apoptosis, and also reduces cyclin D1 expression in breast cancer and lymphoma cell lines. We hypothesized that this dual activity of silvestrol would make it especially effective in malignancies driven by aberrant cyclin D1 expression. Experimental Design: Mantle cell lymphoma (MCL), characterized by elevated cyclin D1, was used as a model to test this approach. The cyclin D/Rb/E2F1 pathway was investigated in vitro using MCL cell lines and primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL. Results: Silvestrol showed low nanomolar potency both in MCL cell lines and primary MCL tumor cells. D-cyclins were depleted with just 10 nmol/L silvestrol at 16 hours, with subsequent reductions of phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As showed in other leukemias, silvestrol caused Mcl-1 depletion followed by mitochondrial depolarization and caspase-dependent apoptosis, effects not related to inhibition of CDK4/6. Silvestrol significantly (P < 0.0001) prolonged survival in a MCL xenograft model without detectable toxicity. Conclusions: These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic strategy in MCL and other malignancies. Clin Cancer Res; 18(17); 4600–11. ©2012 AACR.

Cyclin dependent kinase (CDK) inhibitors, such as flavopiridol, demonstrate significant single-agent activity in chronic lymphocytic leukemia (CLL), but the mechanism of action in these nonproliferating cells is unclear. Here we demonstrate that CLL cells undergo autophagy after treatment with therapeutic agents, including fludarabine, CAL-101, and flavopiridol as well as the endoplasmic reticulum (ER) stress-inducing agent thapsigargin. The addition of chloroquine or siRNA against autophagy components enhanced the cytotoxic effects of flavopiridol and thapsigargin, but not the other agents. Similar to thapsigargin, flavopiridol robustly induces a distinct pattern of ER stress in CLL cells that contributes to cell death through IRE1-mediated activation of ASK1 and possibly downstream caspases. Both autophagy and ER stress were documented in tumor cells from CLL patients receiving flavopiridol. Thus, CLL cells undergo autophagy after multiple stimuli, including therapeutic agents, but only with ER stress mediators and CDK inhibitors is autophagy a mechanism of resistance to cell death. These findings collectively demonstrate, for the first time, a novel mechanism of action (ER stress) and drug resistance (autophagy) for CDK inhibitors, such as flavopiridol in CLL, and provide avenues for new therapeutic combination approaches in this disease.

The impact of mutation of the ATM (ataxia telangiectasia mutated) gene in chronic lymphocytic leukemia (CLL) treatment outcome has not been examined. We studied ATM mutations in 73 patients treated with fludarabine and rituximab. ATM gene mutation analysis was performed using temperature gradient capillary electrophoresis. The impact of detected variants on overall survival (OS) and progression-free survival (PFS) was tested with proportional hazards models. None of the 73 patients demonstrated truncating ATM mutations; 17 (23%, 95% confidence interval 14 – 35%) had non-silent variants (ATM-NSVs), including 13 known ATM polymorphisms and four missense variants. ATM-NSVs were not significantly associated with any baseline characteristics including immunoglobulin heavy chain variable gene (IGVH) status. In multivariable models, no significant differences in complete response (p = 0.70), PFS (p = 0.59) or OS (p = 0.13) were observed. Our data indicate that truncating ATM mutations are rare in patients with CLL. Furthermore, in this dataset, these non-silent variants had limited impact on PFS and OS.

The nuclear export receptor, Exportin 1 (XPO1), mediates transport of growth-regulatory proteins, including tumor suppressors, and is overactive in many cancers, including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and aggressive lymphomas. Oral selective inhibitor of nuclear export (SINE) compounds that block XPO1 function were recently identified and hold promise as a new therapeutic paradigm in many neoplasms. One of these compounds, KPT-330 (selinexor), has made progress in Phase I/II clinical trials, but systemic toxicities limit its administration to twice-per-week and requiring supportive care. We designed a new generation SINE compound, KPT-8602, with a similar mechanism of XPO1 inhibition and potency but considerably improved tolerability. Efficacy of KPT-8602 was evaluated in preclinical animal models of hematological malignancies, including CLL and AML. KPT-8602 shows similar in vitro potency compared with KPT-330 but lower central nervous system penetration, which resulted in enhanced tolerability, even when dosed daily, and improved survival in CLL and AML murine models compared with KPT-330. KPT-8602 is a promising compound for further development in hematological malignancies and other cancers in which upregulation of XPO1 is seen. The wider therapeutic window of KPT-8602 may also allow increased on-target efficacy leading to even more efficacious combinations with other targeted anticancer therapies.