Upregulated JAK/STAT Signaling Represents a Major Mode of Resistance to HDAC Inhibition In Lymphoma and Provides a Rationale for Novel Combination Therapy

Abstract Abstract 434 Background Histone deacetylase inhibitors (HDACis) have demonstrated significant clinical activity in hematologic malignancies; however, single agent response rates have ranged between 20–50% with the duration of response often measured in months, suggesting that drug resistance is a major mode of failure. The pathways through which these agents work and the means by which tumors develop resistance to them are poorly understood. Combination therapy targeting multiple oncogenic pathways holds the promise to improve upon both the depth and durability of these responses. We investigated the mechanisms of inherent and acquired resistance to HDACis in a broad range of lymphomas. By detailing the molecular pathways implicated in resistance to HDACi, we sought to identify novel combinations of compounds that could overcome potential mechanisms that confer resistance. Methods and Results We tested two separate HDACis, LBH589 and SAHA in 51 cell lines representing a wide range of lymphomas including Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma, and Hodgkin lymphoma. Gene expression array data was generated for all these cell lines. We then identified genes that were significantly associated with resistance to both LBH589 and SAHA (p<.01) and applied hierarchical clustering to identify the functional significance of these genes. Histology was not predictive of sensitivity to either HDACi. These data were then analyzed using gene set enrichment to identify known molecular pathways associated with resistance. Activation of JAK/STAT signaling was found to be a major determinant of resistance among the cell lines that were relatively resistant to HDACi. (P<0.001, FDR <.25). To determine whether these genes that we found to be associated with resistance reflected potential mechanisms of acquired resistance to HDACi therapy, we separately engineered resistance to LBH589 and SAHA in three DLBCL cell lines (LY3, BJAB, Farage) through incremental dose escalation over a period of up to 6 months. Each of these three cell lines demonstrated sustained growth at drug concentrations that were at or above their original IC50. Each of these cell lines were then exposed to the other HDACi and tested for cross resistance. In each case, the cell lines demonstrated complete cross-resistance to the other drug. We then profiled the gene expression of these cell lines that had acquired resistance. Similar to our previous results, these cell lines demonstrated increased signaling through the JAK/STAT pathway, suggesting that mechanisms of inherent and acquired resistance are similar. We therefore reasoned that combining HDAC and JAK inhibition may overcome both inherent and acquired resistance. To investigate this hypothesis, we tested LBH589 and INCB018424, a JAK1/2 inhibitor, alone and in combination in the LY3, TMD-8, U2932, and BJAB cell lines. While INCB018424 demonstrated no single agent cytotoxicity, it yielded a high degree of synergy when combined with LBH589 with the combination index computed by the Chou-Talalay method ranging from .19 to .9. Conclusion HDACis show single agent activity in the treatment of a number of hematologic malignancies, however most patients develop resistance to these drugs after relatively short-lived remissions. Thus, the greatest promise of these drugs may lie in combination with other agents that target molecular pathways that underlie resistance to these drugs. Using gene expression profiling of a broad range of tumor types and sensitivity to HDACis we were able to identify activation of the JAK/STAT pathway as a common feature of inherent and acquired resistance to HDACis. We combined the JAK1/2 inhibitor INCB018424 with LBH589 and demonstrated a high degree of synergy. As the number of small molecule inhibitors with clinical activity increases, the need to identify rational preclinical combinations becomes greater. Pairing gene expression profiling and resistant cell lines is a promising approach to the selection of combinations likely to maximize clinical benefit while limiting toxicity. Disclosures: No relevant conflicts of interest to declare.

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The Combination Of Hypomethylating Agents and Histone Deacetylase Inhibitors(HDACi) Are Synergistically Cytotoxic and Reverse The Malignant Phenotype In Preclinical Models Of T-Cell Lymphoma

Blood ◽

10.1182/blood.v122.21.646.646 ◽

2013 ◽

Vol 122 (21) ◽

pp. 646-646 ◽

Cited By ~ 3

Author(s):

Owen A. O'Connor ◽

Enrica Marchi ◽

Kelly Zullo ◽

Luigi Scotto ◽

Jennifer E. Amengual ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Board Of Directors ◽

Cell Lines ◽

Single Agent ◽

Hematologic Malignancies ◽

Advisory Committees ◽

Methylation Array ◽

Simultaneous Exposure ◽

The Mean

Abstract Both HDAC inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) are known to influence global expression patterns in hematologic malignancies. Little is known about the combination of these two drug classes in lymphoid malignancies. HDACIs have marked single agent activity in the T- cell lymphomas (TCL), although the mechanism of action is not well defined. DNMTIs affect cytosine methylation of genomic DNA and have activity mainly restricted to the myeloid derived hematologic malignancies. The single agent efficacy and synergistic interaction of a panel of HDACIs (panobinostat, belinostat, romidepsin and vorinostat) and DNMTIs (decitabine (DEC), 5-azacytadine (5-AZA)) was evaluated in models of TCL. The molecular basis for the synergistic effect of HDACIs and DNMTIs was evaluated by gene expression profiling (GEP) and CpG methylation CTCL. Single agent concentration and time effect relationships were generated for 2 CTCL (HH, H9) and 2 T-ALL (P12, PF382) cell lines. Romidepsin and belinostat were the most potent HDACIs with the mean 48 hour IC50 of 8.8 nM (range 1.7-2.7 nM) and 85 nM (range 36-136 nM), respectively. Cell viability was not affected by treatment with DEC or 5-AZA at 24 and 48 hours at concentrations as high as 20 μM. Reduction in viability was first demonstrated after 72 hours of exposure to DEC, with the mean IC50 of 14.8 μM (range 0.4 μM- >20uM). Simultaneous exposure of combinations of DEC plus romidepsin or DEC plus belinostat at their IC10, IC20, and IC50 produced marked synergy in all TCL derived cell lines. Simultaneous exposure of DEC plus romidepsin demonstrated the deepest synergy at 72 hours with synergy coefficients in the range of 0.3. Cells treated with the combination of DEC plus romidepsin also demonstrated significant induction of apoptosis as evaluated by annexinV/propridium iodide via FACS analysis and an increase in acetylated histone 3 by immunoblot. The in vivo activity of the combination of DEC plus belinostat was investigated in a xenograft model of CTCL using HH, the most resistant TCL derived cell line. Mice were treated with DEC 1.5 mg/kg (day 29, 33, 35, 37, 39, 41, 43) and/or belinostat 100 mg/kg (day 29-day 47). The combination mouse cohort demonstrated statistically significant tumor growth delay compared to DEC alone (p=0.002) and belinostat alone (p=0.001). The interaction of DEC and romidepsin was analyzed by GEP and methylation array. Interestingly, the baseline malignant phenotype seen in the CTCL cell-lines was reversed. A significant down-regulation of genes involved in biosynthetic pathways including protein and lipid synthesis, and a significant up-regulation of genes responsible for cell cycle arrest were seen. The vast majority (114/138; 92%) of genes modulated by the single agents were similarly modulated by the combination. However, the latter induced a further significant change in the transcriptome, affecting an additional 390 genes. Similarly, methylation array data was analyzed following treatment of these drugs alone and in combination. DEC induced de-methylation of 190 different gene regions corresponding to 175 genes and an additional 335 loci. Interestingly, when combined with romidepsin the number of demethylated gene regions decreased to 85 corresponding to 79 genes, 78 of which were common with DEC and 148 additional loci. The comparison of gene expression and methylation demonstrated a significant inverse relationship (R2 = 0.657) with genes found to be differentially expressed in GEP and methylation analysis. (Figure 1)Figure 1Summary of gene expression and methylation analysis.Figure 1. Summary of gene expression and methylation analysis. These data support the observation that DNMTIs in combination with HDACIs produces significant synergistic activity in models of TCL. Further evaluation of the mechanism of action with DNMTIs in combination with HDACIs is ongoing, and a clinical trial of the combination is now open. Disclosures: O'Connor: Celgene Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Allos Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Off Label Use: Hypomethylating Agents in T-cell lymphoma. Amengual:Acetylon Pharmacueticals, INC: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

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Tipifarnib Is Cytotoxic and a Drug Transporter Inhibiting Chemosensitizer in Hodgkin Lymphoma Tumor Cells.

Blood ◽

10.1182/blood.v110.11.1386.1386 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1386-1386

Author(s):

Boris Boll ◽

Anna Lang ◽

Peer Langendorf ◽

Hinrich P. Hansen ◽

Elke P. von Strandmann ◽

...

Keyword(s):

Hodgkin Lymphoma ◽

Cell Lines ◽

Single Agent ◽

Annexin V ◽

Hematologic Malignancies ◽

Clinical Activity ◽

Drug Transporter ◽

Toxicity Profile ◽

Combination Drug ◽

Index Method

Abstract INTRODUCTION: Patients with Hodgkin lymphoma (HL) still suffer from late toxicities and insufficient treatment of relapses. New therapies should therefore be established. Tipifarnib is a farnesyl transferase inhibitor with an excellent toxicity profile and clinical activity in hematologic malignancies. Recently, tipifarnib has also been shown to potentiate the cytotoxicity of anthracyclines in leukemia cells via the inhibition of the multidrug resistance transporter P-glycoprotein. To date, nothing is known about a functional role of drug resistance transporters or the effects of tipifarnib in HL. METHODS: To test the anti-tumor effects of tipifarnib in HL cell lines, tipifarnib was evaluated in the XTT cytotoxicity assay and the Annexin V binding assay. The combination efficacy of tipifarnib with the anthracycline doxorubicine was monitored using the Chou and Talalay combination index method. Flow cytometry was applied to assess the effects of tipifarnib on drug transporter mediated anthracycline efflux. RESULTS: First, tipifarnib displayed high single agent toxicity in HL cell lines with an average IC50 of < 0.1 μM. Second, the combination of tipifarnib and doxorubicine was highly synergistic at clinically relevant concentrations (1–3 μM and 0.02–0.2 μM, respectively). Third, measurement of residual doxorubicine after incubation with doxorubicine and tipifarnib indicated a strong inhibition of doxorubicine efflux by tipifarnib suggesting a mechanism for the synergy of the two drugs. CONCLUSIONS: Since tipifarnib displays high activity in a panel of HL cell lines, the clinical evaluation of tipifarnib in relapsed/refractory patients as single agent is warranted. Interestingly, tipifarnib exhibits a dual targeting mechanism in HL cells: Potent cytotoxicity as single agent and drug transporter dependent chemosensitization leading to a strong synergy with doxorubicine. The synergistic combinations of tipifarnib with doxorubicine correspond to plasma levels of the two drugs in cancer patients. Doxorubicine is one of the most effective but also most toxic drugs in the standard HL polychemotherapies whereas tipifarnib has a very favourable toxicity profile. Consequently, tipifarnib should be evaluated as a combination drug for HL polychemotherapy to save on doxorubicine dose and to lower acute and long-term toxicities. Tipifarnib might also be examined as a chemosensitizer for the combination therapy of patients with refractory disease.

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SL-401, a Targeted Therapy Directed to the Interleukin-3 Receptor (CD123), and SL-801, a Reversible Inhibitor of Exportin-1 (XPO1), Display Synergistic Anti-Tumor Activity Against Hematologic Malignancies in Vitro

Blood ◽

10.1182/blood.v128.22.4724.4724 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4724-4724 ◽

Cited By ~ 1

Author(s):

John Gionco ◽

Janice Chen ◽

Ross Lindsay ◽

Vince Macri ◽

Christopher L. Brooks

Keyword(s):

Cell Line ◽

Cell Lines ◽

Single Agent ◽

Hematologic Malignancies ◽

Clinical Activity ◽

Employment Equity ◽

Equity Ownership ◽

Rpmi 8226 ◽

Anti Tumor Activity

Abstract Background: Novel combination therapies have shown success in combating tumor heterogeneity and drug resistance. SL-401 is a targeted therapy directed to the interleukin-3 receptor (CD123), which is overexpressed on numerous hematologic malignancies. SL-401 has demonstrated high single agent response rates in an ongoing Phase 2 trial of blastic plasmacytoid dendritic cell neoplasm (BPDCN) and is also being evaluated in the clinic for additional cancers, including acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs) as a single agent, and multiple myeloma (MM) in combination with other agents. While SL-401 has demonstrated robust single agent clinical activity in patients with BPDCN, its unique mechanism of action and non-overlapping side effect profile with other agents may lend itself to combination therapy as well. Another class of drugs that has demonstrated clinical activity against several hematologic and solid malignancies is Exportin-1 (XPO1) inhibitors. SL-801 is a novel oral small molecule that reversibly inhibits XPO1 and has shown potent in vitro and in vivo anti-tumor activity against a broad range of hematologic and solid malignancies. SL-801 is currently being evaluated in a Phase 1 trial of patients with advanced solid tumors, and a Phase 1 trial in advanced hematologic cancers is planned. Here, we investigated the in vitro effect of combination treatment of SL-401 and SL-801 against cell lines of chronic myeloid leukemia (CML), AML, MM, and Hodgkin's lymphoma (HL). Methods: The human K562 CML cell line, MV4-11 AML cell line, RPMI-8226 MM cell line, and L-428 HL cell line were treated with varying concentrations of SL-401 and SL-801 alone or in combination for 48 hours. Cell viability was assessed by the CellTiter Glo in vitro cytotoxicity assay. Combination index (CI) values were calculated using CompuSyn software by the method of Chou and Talalay, and treatment was considered to be synergistic when CI < 1. Caspase activation was measured using the Caspase-Glo 3/7 assay, and lactate dehydrogenase (LDH) release was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay. Results: As single agents, SL-401 and SL-801 demonstrated anti-tumor activity in all four cell lines tested. MV4-11 cells were the most sensitive to both drugs, with an IC50 of 34 pM for SL-401 and 21 nM for SL-801. In the other cell lines, the IC50s for SL-401 were 17 nM in K562 cells, 25 nM in RPMI-8226 cells, and 100 nM in L-428 cells, and the IC50s for SL-801 were 99 nM in K562 cells, 51 nM in RPMI-8226 cells, and 494 nM in L-428 cells. When combined with each other, SL-401 and SL-801 potently inhibited cell growth in all cell lines, and CI calculations indicated that the interaction between the two drugs was synergistic at most dose combinations. Notably, CI values < 0.3 were observed in MV4-11 and L-428 cells, indicative of strong synergy. Consistent with these observations, the combination of SL-401 and SL-801 also induced higher levels of caspase activation and LDH release in MV4-11 and L-428 cells than either drug alone. Conclusion: These findings demonstrate that SL-401 and SL-801, when combined, act synergistically in their in vitro anti-tumor activity against CML, AML, MM, and HL cells. Investigations into the molecular mechanisms underlying the observed synergy are in progress. These promising results provide rationale for further development of SL-401 and SL-801 combination therapy in the treatment of a broad range of hematologic malignancies. Disclosures Gionco: Stemline Therapeutics, Inc.: Employment. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Lindsay:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Macri:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

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Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02093-4 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Tamara Rothenburger ◽

Dominique Thomas ◽

Yannick Schreiber ◽

Paul R. Wratil ◽

Tamara Pflantz ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cell Lines ◽

Nucleoside Analogue ◽

Acquired Resistance ◽

Nucleoside Analogues ◽

Cross Resistance ◽

Enzymatic Assays ◽

Anti Cancer ◽

Acute Myeloid

Abstract Background SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1. Methods CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. Results Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. Conclusion Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

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Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

Cells ◽

10.3390/cells9010142 ◽

2020 ◽

Vol 9 (1) ◽

pp. 142 ◽

Cited By ~ 11

Author(s):

Mariusz L. Hartman ◽

Malgorzata Sztiller-Sikorska ◽

Anna Gajos-Michniewicz ◽

Malgorzata Czyz

Keyword(s):

Phenotypic Plasticity ◽

Cell Lines ◽

Mapk Pathway ◽

Acquired Resistance ◽

Resistance Mechanisms ◽

Genetic Alterations ◽

Resistant Cell ◽

Cross Resistance ◽

Preclinical Model ◽

Development Of Resistance

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines. There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.

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Characterization of Bortezomib Resistance in Multiple Myeloma Cell Lines

Blood ◽

10.1182/blood.v112.11.2639.2639 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2639-2639

Author(s):

Silvia CW Ling ◽

Angela M Nikolic ◽

Ammira Al-Shabeeb ◽

Edwin Lau ◽

Phoebe Joy Ho ◽

...

Keyword(s):

Cell Lines ◽

De Novo ◽

Single Agent ◽

Active Form ◽

Myeloma Cell ◽

Cross Resistance ◽

Parent Cell ◽

Mrna Levels ◽

Adherent Growth

Abstract Bortezomib is the first proteasome inhibitor in clinical use. It is particularly effective in myeloma compared with other cancers; as a single agent, its response rate in relapsed myeloma is about 40%. Nevertheless, a large proportion of patients are primarily resistant and acquired bortezomib resistance is also an emerging issue. The mechanism of bortezomib resistance in myeloma remains elusive. We have previously shown that bortezomib sensitivity correlates inversely with the levels of XBP-1 mRNA in vitro and in myeloma patients. XBP-1 is a major regulator of the unfolded protein response (UPR), which is essential for immunoglobulin folding and assembly, and plasma cell development. Previous studies suggested that immunoglobulin production sensitises myeloma cell lines to proteasome inhibitors (S Meister, et al., 2007, Cancer Res 67:1783). We hypothesize that dependence on XBP-1 and the UPR mediate sensitivity to bortezomib. The goal was to develop bortezomib resistant myeloma cell lines, study their phenotype, characterise the mechanism of resistance and, in particular, study the changes in the UPR. Bortezomib resistant sublines (9 in total) were derived from 4 myeloma cell lines (KMS-11, H929, U266 and OPM2), by long term exposure to bortezomib. The fold resistance varies from 3 to 12 and is stable for at least 4 passages without bortezomib. During the evolution of resistance, the myelomas all adopted adherent growth, independent of the morphology of the parent cell lines. When adherent versions of the parent cell lines were derived without exposure to bortezomib, they were, surprisingly, resistant to bortezomib. All bortezomib-resistant myeloma sublines downregulated XBP-1 mRNA levels and the reduction was correlated with the fold resistance. Moreover, splicing of XBP-1 mRNA to the active form was also reduced. The adherent versions of the parent cell lines that showed de novo resistance to bortezomib also had lower levels of XBP-1. Bortezomib normally induces XBP-1 expression and splicing acutely within 12–24 hours (AH Lee, et al., 2003. PNAS 100: 9946) but the resistant sublines were less responsive than the sensitive parents in this respect. Protein levels of other components of the UPR were evaluated by immunoblotting; BiP, phosphorylated-eIF2α, ATF6α and p58IPK were found to be unchanged in the resistant sublines. Cytoplasmic and secreted immunoglobulins were assayed by immunoblotting and ELISA respectively but no differences between the parent and resistant sublines were observed. In a small cohort of relapsed/refractory patients whose bone marrow biopsy was collected prior to bortezomib treatment, the quantity of intracytoplasmic light chain in the myeloma cells, as detected by flow cytometry, did not predict clinical response. The bortezomib-resistant myeloma sublines showed significant cross-resistance to doxorubicin and, to a lesser extent, vincristine, but none to melphalan. The cross-resistance is associated with adherent growth, as it was also observed in the adherent versions of the parent cell lines which were never exposed to bortezomib. Moreover, functional analysis of the multi-drug transporter activity, by drug accumulation assay, showed no difference between parent and resistant sub-lines. These results suggest that resistance to bortezomib is associated with downregulation of the IRE-XBP-1 pathway, but not necessarily other components of the UPR, consistent with observations by others concerning BiP (DT Rutkowski, et al., 2006. Plos Biology 4, e374). The association between adherence, bortezomib resistance and low XBP-1 requires further study.

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Combination of Sorafenib, Idarubicin, and Cytarabine Has a High Response Rate in Patients with Newly Diagnosed Acute Myeloid Leukemia (AML) Younger Than 65 Years

Blood ◽

10.1182/blood.v112.11.768.768 ◽

2008 ◽

Vol 112 (11) ◽

pp. 768-768 ◽

Cited By ~ 3

Author(s):

Farhad Ravandi ◽

Jorge Cortes ◽

Stefan Faderl ◽

Guillermo Garcia-Manero ◽

Susan O’Brien ◽

...

Keyword(s):

Flow Cytometry ◽

Phase I ◽

Cell Lines ◽

Phase Ii ◽

Kinase Inhibitor ◽

Single Agent ◽

High Response Rate ◽

Clinical Activity ◽

Mutation Burden

Abstract Background: Sorafenib is an orally active multi-kinase inhibitor with potent activity against the Raf/ERK/MEK pathway, VEGFR, PDGFR-β, and c-KIT. In vitro, it has growth-inhibitory effects in several AML cell lines with or without constitutive activation of ERK signaling. Sorafenib selectively induces cell growth arrest and apoptosis in FLT3-mutant human AML cell lines at nM concentrations. In a phase I study of single agent sorafenib in patients (pts) with AML escalating doses were well tolerated with no myelosuppression and with significant clinical activity predominantly (but not exclusively) in FLT3 mutated pts. Methods: This study was conducted to determine the tolerability and efficacy of combination of sorafenib with cytarabine 1.5 g/m2 iv over 24 hours daily × 4 (× 3 for pts over 60) and idarubicin 12 mg/m2 iv daily × 3. In the phase I portion of study, pts with relapsed AML were treated with escalating doses of sorafenib po (400 mg qod, 400 mg daily and 400 mg bid) for 7 days during induction, and 400 mg bid was established as a safe dose for phase II evaluation. Pts achieving CR receive up to 5 courses of consolidation with idarubicin 8 mg/m2 iv daily × 2 and cytarabine 0.75 g/m2 iv daily × 3 in addition to continuous sorafenib 400 mg po bid for up to 28 days per cycle. Maintenance with sorafenib 400 mg bid would continue for up to a year after consolidation. Results: Ten pts (median age 34 years, range 21–58) with relapsed AML (median prior therapy 2, range 1–6) were treated on the phase I portion. Seven had FLT3-ITD mutation (5 with high mutation burden, 2 with low), and 3 were negative. Four achieved CR, and 6 failed. In the phase II portion, 30 pts (including 8 with FLT3-ITD and 2 with FLT3-TKD) have been treated. Median age is 53 years (range 18 – 65) Cytogenetics were diploid in 13, +8 in 3, −5/−7 in 3, t(9;11) in 1, miscellaneous in 6, and unavailable in 4. The median presentation WBC was 4.6 × 109/L (range 1.5 –122.7 × 109/L). FLT3 mutation burden was low in blasts from 4 pts, and high in 6). Five pts were FLT3-ITD+/NPM1-. Among 25 evaluable pts, 22 (88%) have achieved CR (n=19), or CRi (n=3); 1 achieved PR, 1 died at induction from pneumonia, 1 was resistant; 5 pts are too early. The regimen is well tolerated and grade 3 adverse events thought to be possibly related to the study combination have included elevation of transaminases (3), hyperbilirubinemia (4), small bowel obstruction (1), diarrhea (2), rash (2), pericarditis (1), elevated creatinine (1), and atrial fibrillation (1). Median follow-up is 8 weeks (range, 1 – 28) with the probability of survival at 6 months of 87%; 2 pts have relapsed with CR durations of 2 and 3 months. Samples from 8 pts were studied prior to and 24–48 hours post sorafenib administration, and prior to chemotherapy. In six pts (75%), sorafenib alone induced apoptosis in peripheral blood blasts and in CD33/CD34 positive leukemia progenitor cells as determined by flow cytometry. Expression of phospho-ERK (pERK) was detectable by flow cytometry in 5 out of 7 samples tested at baseline; 24-hour exposure to sorafenib resulted in >50% downregulation of pERK in 3 of the 5 samples. Plasma inhibitory assay was performed using day 7 samples from 10 pts; mutant FLT3 was suppressed by all samples with 5-fold more potent suppression against mutant versus wildtype FLT3. Conclusions: Combination of sorafenib with idarubicin and cytarabine is safe and has a high CR rate in frontline therapy of younger pts with AML. Correlative studies confirm potent activity of sorafenib against ERK and FLT3 signaling.

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Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e13569 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e13569-e13569

Author(s):

Enrica Marchi ◽

Matko Kalac ◽

Danielle Bongero ◽

Christine McIntosh ◽

Laura K Fogli ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Cell Lines ◽

Molecular Level ◽

Cell Lymphoma ◽

Single Agent ◽

T Cell Lymphoma ◽

Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

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CDK9 Inhibition Reverses Resistance to ABT-199 (GDC-0199) By Down-Regulating MCL-1

Blood ◽

10.1182/blood.v124.21.2161.2161 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2161-2161 ◽

Cited By ~ 1

Author(s):

Jun Chen ◽

Sha Jin ◽

Paul Tapang ◽

Stephen K Tahir ◽

Morey Smith ◽

...

Keyword(s):

Clin Oncol ◽

Cell Lines ◽

Small Molecule ◽

Hodgkin’S Lymphoma ◽

Acquired Resistance ◽

Hematologic Malignancies ◽

Hodgkin's Lymphoma ◽

Employment Equity ◽

Non Hodgkin's Lymphoma ◽

Equity Ownership

Abstract All authors are employees of AbbVie and participated in the design, conduct, and interpretation of these studies. AbbVie and Genentech provided financial support for these studies and participated in the review and approval of this publication. The BCL-2-selective inhibitor ABT-199 has demonstrated efficacy in numerous preclinical models of hematologic malignancies without causing thrombocytopenia, a dose-limiting toxicity associated with the BCL-2/BCL-XL inhibitor navitoclax (Souers et al. 2013. Nat. Med. 19, 202-208). ABT-199 has also demonstrated clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (Seymour et al. 2014. J. Clin. Oncol. 32, 448s; Davids et al. 2014. J. Clin. Oncol. 32, 544s). Despite these encouraging early clinical data, some subjects do not respond to ABT-199 or progress while on treatment. Pre-clinical models indicate that both intrinsic and acquired resistance may be a consequence of MCL-1 expression. Consequently, we have explored potent and selective small molecule inhibitors of CDK9, a kinase known to maintain the expression of MCL-1 through its role in p-TEFb-mediated transcription. Inhibition of CDK9 resulted in the rapid loss in RNA polymerase II phosphorylation (Serine 5) and MCL-1 expression that was closely followed by the induction of apoptosis in MCL-1-dependent cell lines, a cellular response that could be rescued by overexpression of BCL-2. Substantial synergy was observed between CDK9 inhibitors and ABT-199 in a number of hematologic cell lines with intrinsic or acquired resistance to ABT-199. Direct inhibition of MCL-1 with the small molecule BH3 mimetic A-1210477 was also highly synergistic with ABT-199, further validating the utility of co-inhibiting MCL-1 and BCL-2 function simultaneously in ABT-199 resistant tumors. Importantly, the CDK9 inhibitor-ABT-199 combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in xenograft models of non-Hodgkin’s lymphoma (NHL) and acute myelogenous leukemia (AML). These data indicate that CDK9 inhibitors may be highly efficacious when used in combination with ABT-199 for the treatment of hematologic malignancies. Disclosures Chen: Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Tapang:abbvie: Employment, Equity Ownership. Tahir:abbvie: Employment, Equity Ownership. Smith:abbvie: Employment, Equity Ownership. Xue:abbvie: Employment, Equity Ownership. Zhang:abbvie: Employment, Equity Ownership. Gao:abbvie: Employment, Equity Ownership. Tong:abbvie: Employment, Equity Ownership. Clark:abbvie: Employment, Equity Ownership. Ricker:abbvie: Employment, Equity Ownership. Penning:abbvie: Employment, Equity Ownership. Albert:abbvie: Employment, Equity Ownership. Phillips:abbvie: Employment, Equity Ownership. Souers:abbvie: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership.

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Induction of Resistance to Proteasome Inhibition Preferentially Switches Survival Dependence from Bcl-2 to XIAP in Preclinical Models of Waldenstrom Macroglobulinemia: Pre-Clinical Rationale for Early Clinical Sequencing of ABT199

Blood ◽

10.1182/blood.v126.23.4839.4839 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4839-4839

Author(s):

Sharoon Akhtar ◽

Aneel Paulus ◽

Kelara Samuel ◽

Hassan Yousaf ◽

Davitte Cogen ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Conflicts Of Interest ◽

Family Members ◽

Specific Inhibitor ◽

Cross Resistance ◽

Preclinical Models ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Resistant Cells

Abstract Background: The proteasome is an established and druggable target for the treatment of plasma cell-related malignancies including Waldenstrom macroglobulinemia (WM). WM cells as a consequence of high immunoglobulin production and increased B-cell receptor (BCR) mediated proliferation upregulate proteasome activity. Additionally, microenvironmental influence mediated through BCR signaling directly influences Bcl-2 and its BH3 family members, supporting tumor cell survival. Indeed, WM patients derive significant clinical benefit from proteasome-inhibitor (PI) based therapy with agents such as bortezomib and carfilzomib. However, resistance to PI develops over time and for these patients the optimal choice and sequence of therapy has yet to be determined. Using our WM models of PI-resistance we interrogated molecular events within the BCR and Bcl-2 pathways to determine therapeutic potential of targeting these crucial pathway in PI-resistance. Materials & Methods: WM cell lines (BCWM.1 and MWCL-1) and carfilzomib-resistant (CR) subclones (BCWM.1/CR and MWCL-1/CR) were used in experiments. Gene-expression and long-noncoding (LNC) RNA analysis was performed (Arraystar) and validated by real-time PCR. Bortezomib, carfilzomib, ABT199 and ibrutinib were purchased from Sellekchem. Results: To determine the functional impact of BCR and Bcl-2 signaling in PI-resistance, as well as therapeutic sensitivity of PI-resistant cells to their inhibitors (ibrutinib, ABT-199, respectively), we established and characterized WM cell lines resistant to carfilzomib. BCWM.1/CR cells showed approximately 20-fold resistance to carfilzomib (IC50 = 92.75nM) and MWCL-1/CR cells approximately 10-fold resistance (Fig. 1A). Both CR clones also displayed some cross-resistance to bortezomib. Gene expression and LNC-RNA profiling demonstrated several changes between carfilzomib-resistant vs. sensitive WM cells. Analysis of proteasome-related mRNA revealed downregulation of PSMB5, PSMB1, PSMB2 and PSMB8. Similarly, profiling of BCR-associated genes demonstrated decreased expression of several components, including BTK and SPI1. This observation functionally manifested as reduced sensitivity to the BTK-inhibitor ibrutinib, wherein CR cells displayed 1.5 - 2 fold growth resistance to ibrutinib on 72hr MTS assay. Next we examined the expression of Bcl-2 family members in CR cells. Intriguingly, we observed that Bcl-2 and Mcl-1 were significantly downregulated but XIAP (inhibitor of apoptosis) was significantly increased in CR cells vs. wildtype WM cells- both at transcriptional and translational levels. This suggested that upon acquisition of CR, a transcriptional shift towards XIAP occurs to accommodate sustained therapeutic stress from carfilzomib and maintain steady antiapoptotic composure. To test if the PI-resistant cells have moved away from their survival dependence on Bcl-2, we treated CR cells to increasing concentrations of the Bcl-2-specific inhibitor, ABT199, and as anticipated, CR cells displayed reduced apoptosis in presence of ABT199 compared to wildtype WM cells (32% annexin-V staining vs. 50%, respectively) (Fig. 1B). Conclusions: Our study sheds insight into the differential drivers of PI-resistance particularly towards carfilzomib, in WM cells. We demonstrate that acquisition of CR is associated with downregulation of Bcl-2 and Mcl-1, which is countered by upregulation of XIAP- an event that renders CR cells resistant to ABT199 (as it targets only Bcl-2). Likewise, downregulation of BCR-related components in CR cells was associated with reduced sensitivity towards ibrutinib. These observations suggest that acquisition of resistance to PI such as carfilzomib can impact future treatment with agents such as ibrutinib or ABT199. Our preclinical models provide rationale or early sequencing of ibrutinib or ABT199 in therapeutic planning of WM patients prior to induction of PI resistance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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