Chaetocin, a thiodioxopiperazine natural product previously unreported to have anticancer effects, was found to have potent antimyeloma activity in IL-6-dependent and -independent myeloma cell lines in freshly collected sorted and unsorted patient CD138(+) myeloma cells and in vivo. Chaetocin largely spares matched normal CD138(-) patient bone marrow leukocytes, normal B cells, and neoplastic B-CLL (chronic lymphocytic leukemia) cells, indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore, chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone, and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non-cross-resistant to chaetocin. Mechanistically, chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell, its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover, the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but, instead, heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively, chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic.

No abstract

Richter Transformation (RT) develops in CLL as an aggressive, therapy-resistant, diffuse large B cell lymphoma (RT-DLBCL), commonly clonally-related (CLR) to the concomitant CLL. Lack of available pre-clinical human models has hampered the development of novel therapies for RT-DLBCL. Here, we report the profiles of genetic alterations, chromatin accessibility and active enhancers, gene-expressions and anti-lymphoma drug-sensitivity of three newly established, patient-derived, xenograft (PDX) models of RT-DLBCLs, including CLR and clonally-unrelated (CLUR) to concomitant CLL. The CLR and CLUR RT-DLBCL cells display active enhancers, higher single-cell RNA-Seq-determined mRNA, and protein expressions of IRF4, TCF4, and BCL2, as well as increased sensitivity to BET protein inhibitors. CRISPR knockout of IRF4 attenuated c-Myc levels and increased sensitivity to a BET protein inhibitor. Co-treatment with BET inhibitor or BET-PROTAC and ibrutinib or venetoclax exerted synergistic in vitro lethality in the RT-DLBCL cells. Finally, as compared to each agent alone, combination therapy with BET-PROTAC and venetoclax significantly reduced lymphoma burden and improved survival of immune-depleted mice engrafted with CLR-RT-DLBCL. These findings highlight a novel, potentially effective therapy for RT-DLBCL.

There is a clear need to develop novel therapies that would overcome differentiation block and eliminate AML stem/progenitor cells. Genetic and epigenetic dysregulation of enhancers regulates expressions of myeloid lineage transcriptional regulators and their target genes in AML stem/progenitor cells. LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2), which regulates enhancer maintenance and transcription in AML stem/progenitor cells (LSCs). LSD1 is part of the repressor complexes involving HDACs, CoREST or GFI1 that mediate transcriptional repression and differentiation block in AML blast progenitor cells (BPCs). We had previously reported that treatment with the reversible LSD1 inhibitor (LSDi) SP2509 increases the permissive H3K4Me3 mark on the chromatin, associated with induction of p21, p27 and CEBPα levels, as well as of differentiation and loss of viability of AML BPCs (Leukemia. 2014; 28: 2155-64). In the present studies, we further evaluated the anti-AML efficacy of LSD1i-based combination with BET protein inhibitor (BETi). First, we determined that tet-inducible shRNA to KDM1A depleted protein levels of KDM1A, repressed c-Myc, but de-repressed p21, CD11b (ITGAM), CD86 and CEBPα, thereby inhibiting colony growth and modestly inducing lethality in genetically diverse cultured AML cell lines. Following sgRNA-directed, CRISPR/Cas9-mediated gene-editing of LSD1 in AML BPCs, surviving clones exhibited ~50% KDM1A levels and decreased c-Myc and DNMT1 expressions compared to the control AML BPCs. Treatment with either the reversible LSDi, SP2577 (Salarius Pharma), or with the irreversible LSDi ORY-1001, disrupted binding of KDM1A with CoREST. Following LSDi treatment, ATAC-Seq analyses demonstrated significant increase in the accessible chromatin of AML BPCs (represented by gained peaks). Gained ATAC-Seq peaks also involved the chromatin of MED11/13, LY96, CEBPB, RARA, CDKN1C and CD86 genes. ChIP-Seq analysis also showed increased H3K27Ac peaks in the chromatin of CD86, ITGAM, SAMHD1, TET2, MED12 and E2F1, and a reduction of peaks in RUNX1, CDK6, KIT, CTNNB1, HOXB5, FLT3 and MEIS1. RNA-Seq analyses after LSD1i treatment also showed significant perturbations (log2 fold-change >1.25 and p<0.05) in the mRNA expressions, including those of ITGAM, LY96, CD86, SAMHD1, IRF8, APAF1, CDK6, and KIT. Gene set enrichment analysis against Hallmark and Transcription Factor-Target datasets showed positive enrichment of E2F and GFI1 targets, as well as of IL2-STAT5 and TGF-beta signaling, but significant depletion (FDR q-values <0.1) of MYC-targets and genes involved in oxidative phosphorylation. QPCR and Western analyses following LSD1i treatment confirmed significant up regulation of mRNA and protein levels, respectively, of ITGAM, CD86 and LY96 in cultured and primary patient-derived AML BPCs. This was associated with morphologic features of differentiation and inhibition of colony growth in AML cells (OCI-AML5, MOLM13, THP1 and MV4-11) (p < 0.01). We also queried for expression mimickers (EMs) through connectivity mapping of the mRNA signature following LSD1i treatment, utilizing the LINCS1000-CMap analyses. Among the top EM hits were BET protein inhibitors (BETis). Treatment with BETi or BET protein degraders (PROTACs) depleted LSD1 levels in AML BPCs. Utilizing ChIP-Seq data, we also noted that LSD1 promoter is occupied by BET protein BRD4 in the AML cells. Consistent with this, treatment with the BETi OTX015 depleted KDM1A expression in AML cells. Notably, co-treatment with LSDi (SP2577 or ORY-1001) and OTX015 induced synergistic lethality in AML BPCs, including of CD34+, CD38-, Lin- AML stem/progenitor cells (combination indices < 1.0). This was associated with greater depletion of c-Myc, c-Myb and PU.1, but greater induction of p21 and p27. LSD1i or BETi treatment significantly improved survival of the immune-depleted mice (compared to the control mice) engrafted with the AML OCI-AML5 cells or patient-derived xenograft (PDX) models of AML (p < 0.01). Collectively, these findings elucidate the molecular mechanisms and strongly support further in vivo testing and pre-clinical development of LSD1i-based combinations with BETi against AML BPCs. Disclosures Soldi: Beta Cat Pharma: Employment. Han:Beta Cat Pharma: Employment. DiNardo:Agios: Consultancy, Other: Advisory role; Bayer: Other: Advisory role; Celgene: Other: Advisory role; Medimmune: Other: Advisory role; Karyopharm: Other: Advisory role; AbbVie: Consultancy, Other: Advisory role. Kadia:Celgene: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Abbvie: Consultancy; Abbvie: Consultancy; BMS: Research Funding; Jazz: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Research Funding; Novartis: Consultancy. Khoury:Stemline Therapeutics: Research Funding.

Bromodomain extra-terminal (BET) protein (BETP) family of chromatin reader proteins includes BRD4 that binds to acetylated lysine in the active chromatin and transcription factors (TFs) at enhancers and promoters, as well as increases RNA pol II (RNAP2) mediated mRNA transcription of active oncogenes in AML. Disruption of binding and eviction of BRD4 from chromatin results in transcriptional attenuation of pro-growth and pro-survival oncoproteins. These include c-Myc, BCL2, MCL1, CDK6, while inducing p21, p27 and HEXIM1, thereby causing growth inhibition and apoptosis of AML blast progenitor cells (BPCs). Consistent with this, first generation BETP inhibitors (BETi) (e.g., OTX015) have been shown to reduce AML burden and induce clinical remissions, albeit in a minority of patients with AML. Recently, more potent BETis such as ABBV-075 (AbbVie Inc.) have been developed and are being investigated for their clinical efficacy in AML. Venetoclax (ABT-199, AbbVie) and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis in AML BPCs. Treatment with venetoclax alone, and in combinations with other anti-AML agents, is effective in inducing clinical remissions in AML. Here, we interrogated the epigenetic mechanisms underlying transcriptional repression due to BETi treatment, as well as determined the anti-AML activity of co-treatment with BETi and venetoclax or A-1210477 against AML BPCs. ATAC-Seq analysis showed that, in BETi-treated (over untreated control) AML BPCs, BETi treatment induced significantly greater perturbations in the accessible chromatin (number of peaks gained or lost), which were especially enriched for TF-binding sites for RUNX1, c-Myc, GATA2, PU.1 and ERG, especially in the DNA of BCL2, Bcl-xL, MCL1, MYC, BIM, PIM1, CDK6, BRD2/4, HEXIM1, CDKN1A, CEBPA and ITGAM. RNA-Seq analyses, followed by confirmation with QPCR, demonstrated that BETi treatment attenuated expression of MYC, BCL2, Bcl-xL and CDK6, while inducing mRNA expression of HEXIM1 and CDKN1A. BETi (ABBV-075) treatment also dose-dependently reduced protein levels of c-Myc, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A and cleaved PARP levels in AML BPCs. This was associated with dose-dependent (10 to 250 nM) ABBV-075-induced apoptosis of cultured AML cell lines and AML BPCs. Whereas treatment with venetoclax (20 to 200 nM) or A-1210477 (1 to 10 µM) alone also induced apoptosis, co-treatment with ABBV-075 and venetoclax or A-1210477 synergistically induced apoptosis of AML BPCs, including CD34+ patient-derived AML BPCs (combination indices < 1.0). Notably, treatment with venetoclax significantly increased protein expression of MCL1 (p < 0.05), which has been recently reported as a potential mechanism of acquired-adaptive resistance to venetoclax. However, co-treatment with ABBV-075 significantly abrogated venetoclax-induced MCL-1 levels (p < 0.01), which likely contributed to the synergistic anti-AML activity of co-treatment with ABBV-075 and venetoclax against AML BPCs. We next determined the in vivo anti-AML efficacy of ABBV-075 and venetoclax in luciferase-transduced, AML BPCs (MOLM13) and patient-derived xenograft (PDX) models of AML BPCs engrafted in immune depleted (NSG) mice. Compared to treatment with either agent alone, co-treatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell burden (p < 0.001), without inducing toxicity, in AML BPC-engrafted NSG mice. Co-treatment with ABBV-075 and venetoclax also improved the median and overall survival of NSG mice engrafted with MOLM13 AML BPCs (p < 0.0001). Collectively, these pre-clinical findings elucidate the mechanistic rationale, and support evaluation of the clinical efficacy and safety of targeted co-treatment with BETi and BCL2 or MCL1 inhibitor in AML. Disclosures Kadia: Jazz: Consultancy, Research Funding; BMS: Research Funding; Novartis: Consultancy; Abbvie: Consultancy; Celgene: Research Funding; Celgene: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Abbvie: Consultancy; Amgen: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; BMS: Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Research Funding. DiNardo:Karyopharm: Other: Advisory role; Medimmune: Other: Advisory role; Celgene: Other: Advisory role; Bayer: Other: Advisory role; Agios: Consultancy, Other: Advisory role; AbbVie: Consultancy, Other: Advisory role. Khoury:Stemline Therapeutics: Research Funding. Shen:AbbVie Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding.

No abstract

Introduction: Features of multiple myeloma (MM) include a proliferative clonal plasma cell population, bone resorption, and neovascularization. Cytokines and chemokines represent two families of molecules that are capable of propagating and enhancing these disease features. In this study, we have utilized antibody array technology to assess the contributions of cytokines and chemokines to the progression of disease, and evaluated the contribution of stromal cells (SCs) in their production. Methods: Wild-type and IL-1beta transduced KAS 6/1 myeloma cell lines or bone marrow cells isolated from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), and MM were cultured for 48 hrs. Culture supernatants were either analyzed directly, or co-cultured with normal SCs for an additional 48 hrs +/− IL-1 inhibitors, after which the supernatants were removed and analyzed using antibody arrays. SCs alone were also cultured with recombinant IL-1beta +/− IL-1 receptor antagonist (IL-1Ra) to define IL-1 dependent effects. IL-6 and IL-8 ELISAs were utilized to quantify IL-6 and IL-8 levels, and validate antibody array findings. Results: Antibody array analysis of IL-1 effects on stromal cell cultures using recombinant IL-1beta and supernatants from an IL-1beta transduced myeloma cell line demonstrated that stimulation of IL-6, MCP-1 and IL-8 were induced in an IL-1 and stromal cell dependent manner. Although levels of TIMP-2 varied in these cultures, they appeared unrelated to an IL-1 effect. Studies utilizing supernatants from patient bone marrow cells co-cultured with SCs resulted in levels of IL-6, MCP-1, and IL-8 higher than those seen with patient supernatants alone or SC cultures alone. More interestingly, the IL-8 levels appeared to correlate with diagnosis; MGUS samples generated low levels and MM samples stimulated high levels. Furthermore, this stimulation was reduced by the addition of IL-1 inhibitors, demonstrating a dependence on IL-1. To confirm the relationship between diagnosis and IL-8 production, the levels of IL-8 produced by the bone marrow supernatants were quantified directly by ELISA. Correlating with the antibody array data, background production of IL-8 from the cultures of patient cells alone was lower than the corresponding co-culture value. Supernatants from MM patients and a subset of SMM patients stimulated high levels of SC IL-8 secretion in contrast to bone marrow cell supernatants from MGUS patients and most SMM patients. This activity was inhibited by IL-1 inhibitors (see Figure). The IL-8 levels closely parallel the IL-1beta induced IL-6 levels in the same samples. Conclusion: These data indicate that the concentration of IL-8 may be relevant to the pathogenesis of MM. IL-8 production is largely dependent on SCs, and production appears to be at least partially dependent on IL-1 function. IL-8 is a chemokine with activities including chemotaxis of neutrophils, increased vascular permeability and angiogenesis. IL-8 expression has been implicated in multiple tumor types and may play an important role in the stimulation of angiogenesis during the progression from MGUS to active MM. Figure Figure

Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by the expansion of a plasma cell clone that localizes to the bone marrow. Stromal cells residing in the bone marrow respond to signals from MM cells and other cell types by producing cytokines and other proteins that stimulate tumor cell growth, survival, adhesion, migration, and drug resistance. We have examined the proteins produced by stromal cells in response to stimulation by bone marrow from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. Furthermore, we have begun analyzing the properties of one of these proteins, the pro-angiogenic chemokine IL-8, in MM. Methods: Bone marrow aspirates from patients with MGUS (n=3), SMM (n=7), and MM (n=6) were cultured for 48 hours, and the culture supernatants were incubated with stromal cells for an additional 48 hours. Protein levels were analyzed using antibody array and ELISA. Microvessel density (MVD) was determined as a measure of angiogenesis in patient bone marrow samples using CD34 staining. Flow cytometry analysis of MM cell lines and patient bone marrow samples was performed using monoclonal antibodies against IL-8 receptors CXCR1 and CXCR2. Results and Conclusion: We observed a significant increase in stromal cell IL-8 production stimulated by bone marrow cells from patients with active myeloma and a subset of SMM patients (16.67 ± 9.82 ng/ml) in comparison to bone marrow of patients with MGUS and all other SMM patients (0.55 ± 0.17 ng/ml; P=0.0004). Use of an IL-1 inhibitor and recombinant IL-1β demonstrated that IL-8 production was dependent upon IL-1β signaling. Increased BM microvessel density correlated with stimulation of stromal cell IL-8 production (P=0.0005). Furthermore, the majority of MM cell lines (7/9) and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. In addition to its function as a pro-angiogenic factor, IL-8 may directly influence MM cells through its CXCR1 and CXCR2 receptors. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1β induced, correlates with bone marrow angiogenesis, and may influence MM disease via impact upon both the microenvironment and tumor cells.