James M Bogenberger

RNAi screening of the kinome with cytarabine in leukemias

To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

CHK1 and WEE1 inhibition combine synergistically to enhance therapeutic efficacy in acute myeloid leukemia ex vivo.

Novel combinations targeting new molecular vulnerabilities are needed to improve the outcome of patients with acute myeloid leukemia. We recently identified WEE1 kinase as a novel target in leukemias. To identify genes that are synthetically lethal with WEE1 inhibition, we performed a short interfering RNA screen directed against cell cycle and DNA repair genes during concurrent treatment with the WEE1 inhibitor MK1775. CHK1 and ATR, genes encoding two replication checkpoint kinases, were among the genes whose silencing enhanced the effects of WEE1 inhibition most, whereas CDK2 short interfering RNA antagonized MK1775 effects. Building on this observation, we examined the impact of combining MK1775 with selective small molecule inhibitors of CHK1, ATR and cyclin-dependent kinases. The CHK1 inhibitor MK8776 sensitized acute myeloid leukemia cell lines and primary leukemia specimens to MK1775 ex vivo, whereas smaller effects were observed with the MK1775/MK8776 combination in normal myeloid progenitors. The ATR inhibitor VE-821 likewise enhanced the antiproliferative effects of MK1775, whereas the cyclin-dependent kinase inhibitor roscovitine antagonized MK1775. Further studies showed that MK8776 enhanced MK1775-mediated activation of the ATR/CHK1 pathway in acute leukemia cell lines and ex vivo. These results indicate that combined cell cycle checkpoint interference with MK1775/MK8776 warrants further investigation as a potential treatment for acute myeloid leukemia.

Synergistic Targeting of AML Stem/Progenitor Cells With IAP Antagonist Birinapant and Demethylating Agents

BACKGROUND Acute myeloid leukemia (AML) therapy has limited long-term efficacy because patients frequently develop disease relapse because of the inability of standard chemotherapeutic agents to target AML stem/progenitor cells. Here, we identify deregulated apoptotic components in AML stem/progenitor cells and investigate the individual and combinatorial effects of the novel inhibitor of apoptosis (IAP) protein antagonist and second mitochondrial-derived activator of caspases (SMAC) mimetic birinapant and demethylating epigenetic modulators. METHODS Protein expression was measured by reversed-phase protein array in AML patient (n = 511) and normal (n = 21) samples and by western blot in drug-treated cells. The antileukemic activity of birinapant and demethylating agents was assessed in vitro and in an in vivo AML mouse xenograft model (n = 10 mice per group). All statistical tests were two-sided. RESULTS Compared with bulk AML cells, CD34(+)38(-) AML stem/progenitors expressed increased cIAP1 and caspase-8 levels and decreased SMAC levels (one-way analysis of variance followed by Tukeys multiple comparison test, P < .001). Birinapant induced death receptor-/caspase-8-mediated apoptosis in AML cells, including in AML stem/progenitor cells, but not in normal CD34(+) cells. Demethylating agents modulated extrinsic apoptosis pathway components and, when combined with birinapant, were highly synergistic in vitro (combination index < 1), and also more effective in vivo (P < .001, by Student t test, for the median survival of birinapant plus 5-azacytadine vs birinapant alone or vs controls). CONCLUSIONS cIAP1, SMAC, and caspase-8 appear to play a role in AML stem cell survival, and synergistic targeting of these cells with birinapant and demethylating agents shows potential utility in leukemia therapy.

Ex vivo activity of BCL-2 family inhibitors ABT-199 and ABT-737 combined with 5-azacytidine in myeloid malignancies

Novel targeted therapies for the treatment of acute myeloid leukemia (AML) and other advanced myeloid malignancies are urgently needed. Recently we reported results from in vitro studies comparing the BCL-2, BCL-XL and BCL-w inhibitor ABT-737 (preclinical compound with similar inhibitory profile to ABT-263/navitoclax) with the selective BCL-2 inhibitor ABT-199, as single-agent and in combination with 5-azacytidine (5-Aza) in AML-derived cell lines [1]. This study demonstrated greater in vitro potency of ABT-737 in most AML-derived cell lines, as compared to ABT-199, including greater sensitization to 5-Aza. Additionally, we demonstrated that the combination of ABT-737 and 5-Aza exhibits strong synergy in short-term ex vivo cultures of myeloid malignancies, including de novo and secondary AML, myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPNs). Tsao et al. reported synergistic activity between 5-Aza and ABT-737 in AML as well [2]. Recently, Pan et al. published a comparison between single-agent ABT-737 or ABT-263 and ABT-199 in vitro and in animal models, demonstrating potent ABT-199 activity in AML, although ABT-263 was more potent in some primary samples [3]. However, none of these studies directly compared the potential of ABT-737 versus ABT-199 to sensitize the anti-leukemic activity of 5-Aza. Therefore, we directly compared the ability of ABT-737 versus ABT-199 to synergize with 5-Aza in myeloid malignancies using short-term ex vivo cultures of primary AML and MDS/chronic myelomonocytic leukemia (CMML) samples. In primary AML and MDS/CMML samples tested in ex vivo drug dose combination response assays (n = 5), all samples showed synergy, and the synergistic effect with 5-Aza was similar for ABT-199 and ABT-737 [Figures 1(A), 1(C) and 1(D)], making this the first published evidence, to our knowledge, that ABT-199 also synergizes with 5-Aza in a limited number of AML and MDS/CMML samples ex vivo. The level of ex vivo synergy for ABT-737 and 5-Aza was comparable to that previously published [1]. Synergy occurred at low nM doses of ABT-737 and ABT-199, mostly in the range of 40–160 nM for both compounds, doses which most frequently corresponded to maximal synergy. The respective 5-Aza concentrations shown in Figure 1 are 10–15% maximal effective concentration (EC10–15) doses for AML samples and EC10–20 doses for MDS/CMML samples, corresponding to 2.5 µM 5-Aza. Antagonism with 5-Aza was observed at high nanomolar doses (> 640 nM) of both ABT compounds, or with exceedingly high (> 200 µM), clinically irrelevant doses of 5-Aza (data not shown). Figure 1 5-Azacytidine synergy in combination with ABT-199 or ABT-737. 5-Azacytidine was applied to ex vivo cultures as a single dose upon experiment initiation and the readout for synergy analyses with CalcuSyn was relative cell number as measured with Cell Titer-Glo ... We also compared the single-agent activity of ABT-199 versus ABT-737 ex vivo. As highlighted above, we previously showed that, as single agent, ABT-737 was more potent than ABT-199 in vitro. However, consistent with recently published data [3], we find that ex vivo ABT-199 and ABT-737 were more similarly potent in AML samples, with EC50 values < 20 nM for both agents [Figure 1(B) and Tables I and ​andII].II]. Thus, the AML-derived cell line panel we previously evaluated had a greater dependency on BCL-XL and/or BCL-w in vitro than the primary samples we analyzed ex vivo. This is also consistent with our recent observations from BH3 profiling assays. BH3 profiling is a functional assay that uses BH3 peptides to indirectly assess induction of mitochondrial outer membrane permeabilization as a surrogate measurement of apoptosis. The BH3 peptides used are referred to as BH3 metrics when associated with BH3 profiling outputs. Previously, we showed that HRK (specific BH3 binding partner for BCL-XL) was important as a BH3 metric in vitro [4,5]; however, NOXA (MCL-1 specific binding partner) as a BH3 metric more strongly correlated with clinical 5-Aza response [1]. While single-agent EC50 values of ABT-737 and ABT-199 were more similar ex vivo than in vitro ABT-737 was still significantly more potent in some ex vivo samples [Figure 1(B) and Table 1]. Our observations are consistent with a recent report [3] showing that although ABT-199 and ABT-737/263 are often equipotent in ex vivo AML samples, some samples are more sensitive to ABT-737/263, while fewer samples are more sensitive to ABT-199. Th us, BCL-XL and BCL-w may play a more important role in modulating apoptosis in a number of cases of AML. Conversely, BCL-2 and hence ABT-199 may have a greater role in other cases of AML. Collectively, these data also support our previous suggestion, that dual BCL-2 family targeting drugs (i.e. BCL-XL and BCL-2, or BCL-XL and MCL-1) could hold more potential for the treatment of some myeloid neoplasms. However, whether more selective or broader BCL-2 family targeting compounds will have greater activity in myeloid malignancies remains an open question, since oncogenic dependency and compound selectivity influence therapeutic index. For example, the on-target side-effect of thrombocytopenia arising from BCL-XL inhibition by navitoclax has prevented further development in leukemias for now, while ABT-199 is currently being investigated as single-agent in an ongoing trial in patients with AML (ClinicalTrials.gov; NCT01994837). Which agent may be more advantageous, and for which patients, can ultimately only be determined clinically. Table I p-Values associated with single-agent ABT-737 and ABT-199 comparisons in AML samples with diff erential activity*. Table II Characteristics of primary samples testedex vivo. The pre-clinical data presented by our and Dr. Konopleva’s groups is promising with regard to the activity of ABT-737 in combination with 5-Aza. Herein we present the first evidence that ABT-199 also potently synergizes with 5-Aza in primary AML and MDS/CMML samples ex vivo. Based on our preclinical data, it can be suggested that such a combination may also be an effective strategy for patients refractory/resistant to monotherapy with 5-Aza or BCL-2 family inhibitors. Navitoclax may still have a role in some cases of AML where additional anti-apoptotic BCL-2 family members such as BCL-XL or BCL-w are operating, such as in erythroid-differentiated disease with high BCL-XL expression [6,7]. Additionally, MCL-1 is thought to be a critical anti-apoptotic gene in AML [8,9]. Evidence suggesting that 5-Aza can reduce MCL-1 protein levels in primary samples [2] provides further impetus to support 5-Aza in combination with ABT-199 or navitoclax, because neither of these compounds inhibits MCL-1. Several mechanistic and clinical questions remain unanswered regarding monotherapy with BCL-2 family inhibitors, as well as their potential to be combined with 5-Aza. A large body of evidence underlies the theory that a therapeutic index for cytotoxic chemotherapy is, at least in part, a function of apoptotic differences between normal and malignant cells/tissues, termed “apoptotic primedness,” not merely differences in proliferation rate as conventionally thought [10,11]. Thus, it is conceivable that BCL-2 family targeting agents may be effective as single-agent therapy, especially in diseases dependent on BCL-2, such as chronic lymphocytic leukemia, where clinical responses have already been observed [12]. In AML, the contribution of anti-apoptotic proteins BCL-2, BCL-XL and MCL-1 in maintaining leukemogenesis alone or combined is not completely resolved. Furthermore, it is not known whether 5-Aza induces additional pro-apoptotic signals that increase the “apoptotic primedness,” resulting in increased therapeutic benefit in combination with BCL-2 family targeting compounds. It is possible that dual or multiple targeting of BCL-2 family proteins may be therapeutically impractical due to side-effects; however, it is likely that a therapeutic index may even be greater for broader BCL-2 family inhibition given that neoplastic myeloid cells persist at a higher apoptotic threshold, likely mediated by redundant expression of BCL-2 and other anti-apoptotic family members. Evidence that BCL-2 and MCL-1 may be selectively up-regulated in leukemic stem cells compared to normal hematopoietic stem cells also supports this concept [8,9,13]. In summary, we show that ABT-199 compared to ABT-737 can result in similarly potent 5-Aza sensitization in AML and MDS ex vivo. Therefore, clinical trials combining 5-Aza with navitoclax, as we previously proposed, or with ABT-199 for the treatment of advanced myeloid malignancies are warranted.

JAK2 inhibitors in the treatment of myeloproliferative neoplasms

Introduction: Dysregulation of JAK-STAT signaling is a pathogenetic hallmark of myeloproliferative neoplasms (MPNs) arising from several distinct molecular aberrations, including mutations in JAK2, the thrombopoietin receptor (MPL), mutations in negative regulators of JAK-STAT signaling, such as lymphocyte-specific adapter protein (SH2B3), and epigenetic dysregulation as seen with Suppressor of Cytokine Signaling (SOCS) proteins. In addition, growth factor/cytokine stimulatory events activate JAK-STAT signaling independent of mutations. Areas covered: The various mutations and molecular events activating JAK-STAT signaling in MPNs are reviewed. Detailed inhibitory kinase profiles of the currently developed JAK inhibitors are presented. Clinical trial results for currently developed JAK targeting agents are comprehensively summarized. The limitations of JAK-STAT targeting in MPNs, as well as potential rational combination therapies with JAK2 inhibitors, are discussed. Expert opinion: Aberrant JAK-STAT signaling is an underlying theme in the pathogenesis of MPNs. While JAK2 inhibitors are active in JAK2V617F and wild-type JAK2 MPNs, JAK2V617F mutation-specific or JAK2-selective inhibitors may possess unique clinical attributes. Complimentary targeting of parallel pathways operating in MPNs may offer novel therapeutic approaches in combination with JAK inhibition. Understanding the intricacies of JAK-STAT pathway activation, including growth factor/cytokine-driven signaling, will open new avenues for therapeutic intervention at known and novel molecular vulnerabilities of MPNs.

Current outlook on molecular pathogenesis and treatment of myeloproliferative neoplasms

Discovery of the JAK2 V617F mutation in the myeloproliferative neoplasms (MPNs) essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) has stimulated great interest in the underlying molecular mechanisms and treatment of these diseases. Along with acceleration of technologies, novel mutations in genes such as MPL, LNK, and CBL have been discovered that converge on the JAK-STAT pathway. Several additional novel mutations in genes involved in epigenetic regulation of the genome, including TET2, ASXL1, DNMT3A, and IDH1/2, have emerged, in addition to several mutations in cellular splicing machinery. While understanding of the pathogenetic mechanisms of these novel mutations in MPNs has improved, it is still lagging behind the pace of mutation discovery. Concurrent with molecular discoveries, especially with regard to JAK-STAT signaling, therapeutic development has accelerated in recent years. More than ten JAK kinase inhibitors have been advanced into clinical trials. Recently the first JAK2 inhibitor was approved for use in patients with PMF. Most JAK-targeting agents share similar characteristics with regard to clinical benefit, consisting of improvements in splenomegaly, constitutional symptoms, and cytopenias, for example. It remains to be determined if JAK2 inhibitors can considerably impact disease progression and bone marrow histologic features (e.g., fibrosis) or significantly impact the JAK2 allele burden. While JAK2 inhibitors appear to be promising in PV and ET, they need to be compared with standard therapies, such as hydroxyurea or interferon-based therapies. Future clinical development will focus on optimal combination partners and agents that target alternative mechanisms, deepen the response, and achieve molecular remissions.

Genomic analysis of hairy cell leukemia identifies novel recurrent genetic alterations

Classical hairy cell leukemia (cHCL) is characterized by a near 100% frequency of the BRAFV600E mutation, whereas ∼30% of variant HCLs (vHCLs) have MAP2K1 mutations. However, recurrent genetic alterations cooperating with BRAFV600E or MAP2K1 mutations in HCL, as well as those in MAP2K1 wild-type vHCL, are not well defined. We therefore performed deep targeted mutational and copy number analysis of cHCL (n = 53) and vHCL (n = 8). The most common genetic alteration in cHCL apart from BRAFV600E was heterozygous loss of chromosome 7q, the minimally deleted region of which targeted wild-type BRAF, subdividing cHCL into those hemizygous versus heterozygous for the BRAFV600E mutation. In addition to CDKN1B mutations in cHCL, recurrent inactivating mutations in KMT2C (MLL3) were identified in 15% and 25% of cHCLs and vHCLs, respectively. Moreover, 13% of vHCLs harbored predicted activating mutations in CCND3 A change-of-function mutation in the splicing factor U2AF1 was also present in 13% of vHCLs. Genomic analysis of de novo vemurafenib-resistant cHCL identified a novel gain-of-function mutation in IRS1 and losses of NF1 and NF2, each of which contributed to resistance. These data provide further insight into the genetic bases of cHCL and vHCL and mechanisms of RAF inhibitor resistance encountered clinically.

Combined venetoclax and alvocidib in acute myeloid leukemia

More effective treatment options for elderly acute myeloid leukemia (AML) patients are needed as only 25–50% of patients respond to standard-of-care therapies, response duration is typically short, and disease progression is inevitable even with some novel therapies and ongoing clinical trials. Anti-apoptotic BCL-2 family inhibitors, such as venetoclax, are promising therapies for AML. Nonetheless, resistance is emerging. We demonstrate that venetoclax combined with cyclin-dependent kinase (CDK) inhibitor alvocidib is potently synergistic in venetoclax-sensitive and -resistant AML models in vitro, ex vivo and in vivo. Alvocidib decreased MCL-1, and/or increased pro-apoptotic proteins such as BIM or NOXA, often synergistically with venetoclax. Over-expression of BCL-XL diminished synergy, while knock-down of BIM almost entirely abrogated synergy, demonstrating that the synergistic interaction between alvocidib and venetoclax is primarily dependent on intrinsic apoptosis. CDK9 inhibition predominantly mediated venetoclax sensitization, while CDK4/6 inhibition with palbociclib did not potentiate venetoclax activity. Combined, venetoclax and alvocidib modulate the balance of BCL-2 family proteins through complementary, yet variable mechanisms favoring apoptosis, highlighting this combination as a promising therapy for AML or high-risk MDS with the capacity to overcome intrinsic apoptosis mechanisms of resistance. These results support clinical testing of combined venetoclax and alvocidib for the treatment of AML and advanced MDS.

Novel targeted therapy strategies for biliary tract cancers and hepatocellular carcinoma

Worldwide hepatobiliary cancers are the second leading cause of cancer related death. Despite their relevance, hepatobiliary cancers have a paucity of approved systemic therapy options. However, there are a number of emerging therapeutic biomarkers and therapeutic concepts that show promise. In hepatocellular carcinoma, nivolumab appears particularly promising and recently received US FDA approval. In intrahepatic cholangiocarcinoma, therapies targeting FGFR2 and IDH1 and immune checkpoint inhibitors are the furthest along and generating the most excitement. There are additional biomarkers that merit further exploration in hepatobiliary cancers including FGF19, ERRFI1, TERT, BAP1, BRAF, CDKN2A, tumor mutational burden and ERBB2 (HER2/neu). Development of new and innovative therapies would help address the unmet need for effective systemic therapies in advanced and metastatic hepatobiliary cancers.