Yuling Chen

Transport properties of pancreatic cancer describe gemcitabine delivery and response

BACKGROUND The therapeutic resistance of pancreatic ductal adenocarcinoma (PDAC) is partly ascribed to ineffective delivery of chemotherapy to cancer cells. We hypothesized that physical properties at vascular, extracellular, and cellular scales influence delivery of and response to gemcitabine-based therapy. METHODS We developed a method to measure mass transport properties during routine contrast-enhanced CT scans of individual human PDAC tumors. Additionally, we evaluated gemcitabine infusion during PDAC resection in 12 patients, measuring gemcitabine incorporation into tumor DNA and correlating its uptake with human equilibrative nucleoside transporter (hENT1) levels, stromal reaction, and CT-derived mass transport properties. We also studied associations between CT-derived transport properties and clinical outcomes in patients who received preoperative gemcitabine-based chemoradiotherapy for resectable PDAC. RESULTS Transport modeling of 176 CT scans illustrated striking differences in transport properties between normal pancreas and tumor, with a wide array of enhancement profiles. Reflecting the interpatient differences in contrast enhancement, resected tumors exhibited dramatic differences in gemcitabine DNA incorporation, despite similar intravascular pharmacokinetics. Gemcitabine incorporation into tumor DNA was inversely related to CT-derived transport parameters and PDAC stromal score, after accounting for hENT1 levels. Moreover, stromal score directly correlated with CT-derived parameters. Among 110 patients who received preoperative gemcitabine-based chemoradiotherapy, CT-derived parameters correlated with pathological response and survival. CONCLUSION Gemcitabine incorporation into tumor DNA is highly variable and correlates with multiscale transport properties that can be derived from routine CT scans. Furthermore, pretherapy CT-derived properties correlate with clinically relevant endpoints. TRIAL REGISTRATION Clinicaltrials.gov NCT01276613. FUNDING Lustgarten Foundation (989161), Department of Defense (W81XWH-09-1-0212), NIH (U54CA151668, KCA088084).

Homoharringtonine reduced Mcl-1 expression and induced apoptosis in chronic lymphocytic leukemia

Homoharringtonine (HHT) is a plant alkaloid that inhibits the elongation phase of translation that is currently in clinical trials. Because the intrinsically short-lived antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) has been reported to support the survival of chronic lymphocytic leukemia (CLL) cells, we hypothesized that inhibition of protein synthesis by HHT would decrease Mcl-1 expression and induce apoptosis in CLL. In primary CLL cells, HHT induced significant apoptosis independent of the prognostic characteristics of the patients. This was associated with inhibition of translation and decreased Mcl-1 levels in CLL cells. Mcl-1 reduction was evident as early as 2 hours and continued to decrease in the next 6-8 hours, whereas cell death started in 2 hours and continued to increase for 24 hours. Reduction of the Mcl-1 level was due to translation inhibition and proteasome degradation rather than to transcription inhibition or caspase cleavage. HHT and the transcription inhibitor SNS-032 induced synergistic cell killing. Although stromal cells induced Mcl-1 expression and protected CLL cells from the toxicity of fludarabine, this induction was reversed by HHT, which overcame stromal cell-mediated protection. Thus, these results provide a rationale for clinical development of HHT in CLL as single agent or in combinations.

Intra-tumoral heterogeneity of gemcitabine delivery and mass transport in human pancreatic cancer

There is substantial heterogeneity in the clinical behavior of pancreatic cancer and in its response to therapy. Some of this variation may be due to differences in delivery of cytotoxic therapies between patients and within individual tumors. Indeed, in 12 patients with resectable pancreatic cancer, we previously demonstrated wide inter-patient variability in the delivery of gemcitabine as well as in the mass transport properties of tumors as measured by computed tomography (CT) scans. However, the variability of drug delivery and transport properties within pancreatic tumors is currently unknown. Here, we analyzed regional measurements of gemcitabine DNA incorporation in the tumors of the same 12 patients to understand the degree of intra-tumoral heterogeneity of drug delivery. We also developed a volumetric segmentation approach to measure mass transport properties from the CT scans of these patients and tested inter-observer agreement with this new methodology. Our results demonstrate significant heterogeneity of gemcitabine delivery within individual pancreatic tumors and across the patient cohort, with gemcitabine DNA incorporation in the inner portion of the tumors ranging from 38 to 74% of the total. Similarly, the CT-derived mass transport properties of the tumors had a high degree of heterogeneity, ranging from minimal difference to almost 200% difference between inner and outer portions of the tumor. Our quantitative method to derive transport properties from CT scans demonstrated less than 5% difference in gemcitabine prediction at the average CT-derived transport value across observers. These data illustrate significant inter-patient and intra-tumoral heterogeneity in the delivery of gemcitabine, and highlight how this variability can be reproducibly accounted for using principles of mass transport. With further validation as a biophysical marker, transport properties of tumors may be useful in patient selection for therapy and prediction of therapeutic outcome.

Derivatization of agelastatin A leading to bioactive analogs and a trifunctional probe.

(-)-Agelastatin A (AglA, 1), a member of the pyrrole-aminoimidazole marine alkaloid (PAI) family, possesses a unique tetracyclic structure and is one of the most potent anticancer PAIs isolated to date. In efforts to expand the SAR of these agents and delineate sites that tolerate modification while retaining activity, we synthesized several derivatives and tested their anticancer activity. The cytotoxic effects of these derivatives were measured against several cancer cell lines including cervical cancer (HeLa), epidermoid carcinoma (A431), ovarian (Igrov and Ovcar3), osteosarcoma (SJSA1), acute T cell leukemia (A3), epidermoid carcinoma (A431) in addition to primary human chronic lymphocytic leukemia (CLL) cells. New positions for modification of AglA and new substitutions were explored leading to novel derivatives, 14-chloro AglA (3) and 14-methyl AglA (12), that retained activity toward various cancer cell lines with decreased toxicity toward B- and T-cells. The SAR data informed the synthesis of a trifunctional probe bearing an alkyne and a diazirine potentially useful for cellular target identification.

Abstract 343: Targeting SF3B1 mediated splicing control of Mcl-1 in chronic lymphocytic leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Recently, deep sequencing technology identified SF3B1, a gene encoding a splicing factor, is recurrently mutated in chronic lymphocytic leukemia (CLL). Moreover, its mutation was associated with rapid disease progression and resistance to treatment. SF3B1 is a critical component of the spliceosome, leading to the potential of splicing regulation in CLL pathogenesis. However, the mechanism by which SF3B1 mutations affect CLL disease progression is not known. CLL cells are characterized by their resistance to apoptosis, driven mostly by the overexpression of the Bcl-2 family anti-apoptotic proteins. Many members of the family have multiple splicing variants, sometimes encoding proteins with totally opposite functions. For example, the alternatively spliced, shorter variant of the anti-apoptotic protein Mcl-1, Mcl-1S, resembles BH3-only proteins and induces apoptosis. Using herboxidiene, an inhibitor of SF3B1, we explored the activity of SF3B1 in splicing control of Mcl-1 in primary CLL cells. Primers and probes were designed to specifically detect the Mcl-1 pre-mRNA, the anti-apoptotic Mcl-1 and pro-apoptotic Mcl-1S by real-time RT-PCR. We found that inhibition of SF3B1 reduced the level of Mcl-1 in a concentration dependent manner, which was accompanied by the simultaneous surge of Mcl-1S mRNA by over 10 fold. Immunoblots confirmed reduction of Mcl-1L and concurrent increase of Mcl-1S protein. These results indicated a major role of SF3B1 in Mcl-1 splicing control toward the anti-apoptotic variant. In contrast, although also depleting the Mcl-1 mRNA, the Cdk9 inhibitor SNS-032 reduced both the long and short forms of Mcl-1, as well as its pre-mRNA, consistent with its inhibition on transcription. Uridine incorporation measurements showed inhibition of total RNA synthesis by herboxidiene at concentrations above 30 nM, but dramatic reversion of Mcl-1 splicing was observed at 10 nM, which did not affect RNA synthesis, suggesting that the major mechanism of herboxidiene in CLL cells was modification of Mcl-1 splicing. As CLL cells are critically dependent on Mcl-1 for survival, reducing its level induced mitochondrial membrane permeabilization and apoptosis, which was further accelerated by the accumulation of Mcl-1S. Apoptosis occurred in a time- and concentration- dependent manner, with IC50 of less than 100 nM after 24 hr of incubation. Herboxidiene toxicity was selective to CLL B cells, while sparing the normal lymphocytes from healthy donors. Herboxidiene was equally toxic to CLL cells harboring SF3B1 mutations compared to cells carrying the wild-type SF3B1 (n=5 each). Real-time RT-PCR showed similar increase in the ratio of short to long forms of Mcl-1 by herboxidiene in these two groups. Together, these studies identified SF3B1 mediated splicing control as a novel target for CLL therapy. Further investigations are needed to understand the mechanism of SF3B1 mutations on the control of mRNA splicing. Citation Format: Rong Chen, Yuling Chen, Emanuela M. Ghia, Laura Rassenti, Thomas Kipps, Michael J. Keating, William G. Wierda, William Plunkett. Targeting SF3B1 mediated splicing control of Mcl-1 in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 343. doi:10.1158/1538-7445.AM2014-343

Abstract 3905: Strategic combination of the cyclin-dependent kinase inhibitor CYC065 with venetoclax to target anti-apoptotic proteins in chronic lymphocytic leukemia

CYC065 is a cyclin-dependent kinase (Cdk) inhibitor that is highly selective towards Cdk2 and Cdk9. In chronic lymphocytic leukemia (CLL), a disease that is addicted to the over-expression of anti-apoptotic proteins for survival, inhibition of Cdk9 by CYC065 reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription. These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1, but not Bcl-2, and induced apoptosis in CLL cells in vitro. The IC50 for CYC065-induced CLL cell death after a 24-hr incubation was 0.8 μM, a concentration that is achievable in the clinic at tolerated doses. CYC065 killed the CLL cells equally efficiently in the presence or absence of the human stromal cell line, StromaNKtert, and with or without a stimulation condition that mimics the lymphoid tissue microenvironment (anti-IgM, anti-CD-40, IL-4). Venetoclax, which specifically inhibits Bcl-2 function, is approved for treatment of CLL with del(17p); however upregulation of Mcl-1 is associated with resistance to venetoclax in the lymph nodes. Therefore, we hypothesized that the combination of CYC065 with venetoclax would target the parallel mechanisms that promote the survival control in CLL cells, and induce synergistic cell death by apoptosis. A time course study of the single agents showed that under conditions that mimic the lymph node microenvironment, cell death induction by venetoclax required 6-8 hr to reach the plateau of cell killing and maximal killing by CYC065 occurred after 24 hr, consistent with the different mechanisms of action of the two compounds. Following the removal of CYC065 or venetoclax after 4, 8, 12, or 24 hr incubations, there was no evidence for additional cell death after an additional 48 hr in drug-free medium regardless of the duration of drug incubation. Immunoblots showed recovery of RNA pol II phosphorylation, and restored Mcl-1 expression upon washout of CYC065. The reversible action of these compounds has potential implications for clinical scheduling combining these compounds. Median effect analysis indicated that CYC065 and venetoclax combined synergistically in CLL samples with or without 17p deletion. A dose reduction analysis confirmed mutual potentiation of each other when combined. Combination of IC50 concentrations of CYC065 and venetoclax for 24 hr was sufficient to decrease the viability of CLL cells by over 90% in the lymph node mimicking microenvironment. Thus, these data provided rationale for clinical combination of CYC065 and venetoclax in CLL. CYC065 is currently in a Phase I clinical trial in patients with advanced solid tumors (NCT02552953) using an intermittent dosing regimen which causes at least 24 hr Mcl-1 downregulation in patient PBMCs at well tolerated dose levels. Citation Format: Rong Chen, Yuling Chen, Sheelagh Frame, David Blake, William G. Wierda, Daniella Zheleva, William Plunkett. Strategic combination of the cyclin-dependent kinase inhibitor CYC065 with venetoclax to target anti-apoptotic proteins in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3905.

Targeting BRCA1/2 deficient ovarian cancer with CNDAC-based drug combinations

PurposeThe mechanism of action of CNDAC (2′-C-cyano-2′-deoxy-1-β-d-arabino-pentofuranosyl-cytosine) is unique among deoxycytidine analogs because upon incorporation into DNA it causes a single strand break which is converted to a double strand break after DNA replication. This lesion requires homologous recombination (HR) for repair. CNDAC, as the parent nucleoside, DFP10917, and as an oral prodrug, sapacitabine, are undergoing clinical trials for hematological malignancies and solid tumors. The purpose of this study is to investigate the potential of CNDAC for the therapy of ovarian cancer (OC).MethodsDrug sensitivity was evaluated using a clonogenic survival assay. Drug combination effects were quantified by median effect analysis.ResultsOC cells lacking function of the key HR genes, BRCA1 or BRCA2, were more sensitive to CNDAC than corresponding HR proficient cells. The sensitization was associated with greater levels of DNA damage in response to CNDAC at clinically achievable concentrations, manifested as chromosomal aberrations. Three classes of CNDAC-based drug combinations were investigated. First, the PARP1 inhibitors, rucaparib and talazoparib, were selectively synergistic with CNDAC in BRCA1/2 deficient OC cells (combination index < 1) at a relatively low concentration range. Second, cisplatin and oxaliplatin had additive combination effects with CNDAC (combination index ~ 1). Finally, paclitaxel and docetaxel achieved additive cell-killing effects with CNDAC at concentration ranges of the taxanes similar for both BRCA1/2 deficient and proficient OC cells.ConclusionsThis study provides mechanistic rationales for combining CNDAC with PARP inhibitors, platinum compounds and taxanes in ovarian cancer lacking BRCA1/2 function.

Abstract 1765: Creating novel translation inhibitors to target pro-survival proteins in chronic lymphocytic leukemia

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Many aspects of malignant phenotypes are associated with dysregulation of translation control. Due to the fact that the great majority of oncoproteins turnover rapidly, and have an acute need for mRNA translation compared to housekeeping proteins, protein synthesis is a promising avenue to explore for targeted cancer therapy. We propose to target the biological rewiring that is characteristic of B cell malignancies, namely the overexpression of short-lived pro-survival proteins that keep these cells from undergoing apoptosis. We hypothesize that transient inhibition of translation will cause a lethal decrease in those labile pro-survival proteins in CLL cells initiating apoptosis. Further, this will provide selectivity toward the malignancy over normal cells and may prove to be a more general strategy for overcoming drug resistance. The marine natural product pateamine A (PatA) inhibits cap-dependent translation initiation by binding to the eukaryotic initiation factor 4A (eIF4A) and stalls the initiation complex on mRNA. We have synthesized a family of PatA-based small molecules to test their potency for inducing apoptosis in primary CLL cells. Our lead compound, DMDAPatA, is more stable and easier to synthesize than the parent natural product, and demonstrated anti-leukemia activity in primary CLL cells with an IC50 of 2.5 μM after 24 h of incubation. This derivative was equally active in samples from patients with poor prognostic characteristics such as advanced Rai stage, the unmutated status of the IgVH gene, or the over-expression of the ZAP70 protein, suggesting its ability to bypass resistance to conventional CLL therapy. Tritium labeled leucine incorporation measurements confirmed inhibition of translation by DMDAPatA that occurred as soon as 4 h, preceding the full activation of apoptosis. Reduction of the short-lived anti-apoptotic proteins Mcl-1 and XIAP were observed by 4 h, and persisted over 24 h, while the levels of the more stable proteins Bcl-2 and Bcl-XL were not affected. This is consistent with the selective activity of translation inhibitors toward proteins with rapid turnover rates. Diminished levels of Mcl-1 and XIAP reduced the anti-apoptotic capacity such that the pro-apoptotic proteins became more prevalent to initiate cell death. Since DMDAPatA depleted Mcl-1 without affecting Bcl-2, and ABT-199 inhibits Bcl-2 activity but spares Mcl-1, the combination of DMDAPatA and ABT-199 targeted both arms of apoptosis control and killed CLL cells synergistically. Dose reduction index analysis demonstrated mutual potentiation of ABT-199 and DMDAPatA. Thus, these studies provide the first proof-of-mechanism investigations of PatA analogs for CLL therapeutics. DMDAPatA appeared to be highly plasma protein bound compared to PatA. New derivatives are being synthesized to reduce plasma protein binding and increase potency. Citation Format: Rong Chen, Mingzhao Zhu, Yuling Chen, Wesley Skillern, William G. Wierda, Ken Hull, Daniel Romo, William Plunkett. Creating novel translation inhibitors to target pro-survival proteins in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1765. doi:10.1158/1538-7445.AM2015-1765

Abstract 2808: Mechanism of action of the multikinase inhibitor TG02 in chronic lymphocytic leukemia

TG02 is small molecular multikinase inhibitor with a unique inhibition spectrum of therapeutic targets. It potently inhibits Cdk9 and Cdk7, the Cdks involved in transcription regulation, with IC 50 values of 3 nM and 37 nM, respectively. Cdk1 and 2 are inhibited at IC 50 s of 9 and 5 nM. Kinases in other signaling pathways are also targets of TG02, such as JAK2 and FLT3 (IC 50 19 nM for both). It is currently in Phase I clinical trials in hematological malignancies based on its unique target profile and the encouraging preclinical results. In this study, we investigated its activity in an indolent tumor type: chronic lymphocytic leukemia (CLL). CLL is characterized by the dependence on the continuous expression of anti-apoptotic proteins for survival. Our previous studies showed that inhibiting Cdk9 and Cdk7 blocked the activity of RNA polymerase II (RNA Pol II), which resulted in the depletion of the short-lived anti-apoptotic protein Mcl-1 and induction of apoptosis in primary CLL cells in vitro. This action was confirmed during clinical trials of Cdk9 inhibitors. In this study, we first confirmed the activity of TG02 as a Cdk7/9 inhibitor. Our results showed that the phosphorylation of Ser2 sites (Cdk9 substrate) of RNA Pol II C-terminal domain repeat was significantly reduced at 4 hr by 0.5 μM TG02. Greater concentrations were required to inhibit the phosphorylation at Ser5 sites (Cdk7 substrate), consistent with a higher IC 50 for Cdk7. These actions led to a decrease in RNA Pol II mediated transcription, measured by tritiated uridine incorporation. Mcl-1 expression was reduced rapidly at both the mRNA and protein levels. This was associated with loss of mitochondrial membrane potential and induction of apoptosis. To differentiate the targets that contribute to the induction of apoptosis, toxicity of TG02 was compared to SNS-032 (Cdk9 inhibitor), Sunitinib (FLT3 inhibitor) and TG-101348 (JAK2 inhibitor). The pattern of TG-02 induced cell death was similar to that of SNS-032, whereas sunitinib induced rapid necrosis, and TG-101348 was much less potent. Thus, this data indicated that inhibition of Cdk9 played a major role for the toxicity of TG02 in CLL. While in these experiments CLL cells were cultured in RPMI media supplemented with 10% autologous plasma to mimic the in vivo conditions, our experiments showed that human plasma proteins impact on the potency of TG02. Compared to 10% FBS, the IC 50 for cell death at 24 h was about 4-fold and 18-fold greater in 10% and 50% human plasma, respectively, indicating a substantial plasma protein binding for TG02. Finally, BAX/BAK double knockout MEF cells were resistant to TG02, suggesting that TG02 requires BAX/BAK to induce apoptosis, consistent with a mitochondrial pathway of cell death. Taken together, these studies suggested that among the multiple targets of TG02, inhibition of Cdk7/9 and transcriptional down-regulation of Mcl-1 was the major cytotoxic pathway activated by TG02 in CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2808. doi:1538-7445.AM2012-2808

Abstract 4431: A novel derivative of the Cdk inhibitor roscovitine that induces apoptosis in CLL and overcomes stromal cell-mediated protection

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cmpd 5 is a derivative of R-roscovitine (CYC-202, seliciclib) with increased potency that selectively inhibits Cdk2, Cdk5 and Cdk9. In chronic lymphocytic leukemia (CLL) a disease that is addicted to the over-expression of anti-apoptotic proteins for survival, inhibition of Cdk9 by Cmpd 5 reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription. These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1, and induced apoptosis in CLL cells in vitro. Cmpd 5 was about 30 times more potent than its parent compound R-roscovitine with an IC50 of 0.86 µM after a 24-hr incubation. Although cell death was initiated after a 4-hr incubation in a time course study of Cmpd 5-induced apoptosis, the maximum cell death was not reached until 10-12 hr. In addition, it was known that co-culture of CLL cells with the marrow and lymphoid stromal cells may be responsible for resistance to fludarabine therapy. We evaluated Cmpd 5 in overcoming such stromal cell-mediated protection. Cmpd 5 killed the CLL cells similarly in the presence or absence of the human stromal cell line StromaNKtert without toxicity to the stromal cells, whereas the stromal cells clearly protected the CLL cells from the toxicity of fludarabine. Since Mcl-1 is the major target of Cmpd 5 in CLL, and the action of Cmpd 5 relies on the intrinsically rapid turn-over rate of Mcl-1, we compared the protein levels and the half-life of Mcl-1 in CLL cells in the presence or absence of the stromal cell layer. After an overnight incubation of CLL cells with the StromaNKtert cells, there was a 3-4 fold induction of Mcl-1 transcript and protein. However, there was no difference in the Cmpd 5-induced decay rate of either the Mcl-1 mRNA or protein, indicating that stromal cells did not alter the stability of Mcl-1. Therefore, the elevated Mcl-1 level likely indicated increased biosynthesis, rather than enhanced stability. Thus, the activation of the survival pathways and induction of the biosynthesis of Mcl-1 by the stromal cells may protect CLL cells from fludarabine-induced apoptosis but loss of the short-lived anti-apoptotic proteins was not affected, explaining the lack of stromal cell protection for CLL cells from Cmpd 5. Similar time-dependence of cell death induction as well as stromal cell response was also observed with other inhibitors of transcription including flavopiridol, R-roscovitine and Actinomycin D, implying a class effect. Taken together, our data suggested that Cmpd 5 is a promising candidate for clinical development for CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4431.