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Chaperonin containing TCP1 (CCT/TRiC) is a multi-subunit protein folding complex that enables the cancer phenotype to emerge from the mutational landscape that drives oncogenesis. We and others linked increased expression of CCT subunits to advanced tumor stage and invasiveness that inversely correlates with cancer patient outcomes. In this study, we examined the expression of the second CCT subunit, CCT2, using genomic databases of adult and pediatric tumors and normal tissues, and found that it was highly expressed in pediatric cancers, showing a significant difference compared to normal tissues. Histologic staining confirmed that CCT subunits are highly expressed in tumor tissues, which was exemplified in neuroblastoma. Using two neuroblastoma cells, MYCN-amplified, IMR-32 cells, and non-amplified, SK-N-AS cells, we assessed baseline levels for CCT subunits and found expressions comparable to the highly invasive triple-negative breast cancer (TNBC) cell line, MDA-MB-231. Exogenous expression of CCT2 in both SK-N-AS and IMR-32 cells resulted in morphological changes, such as larger cell size and increased adherence, with significant increases in the CCT substrates, actin, and tubulin, as well as increased migration. Depletion of CCT2 reversed these effects and reduced cell viability. We evaluated CCT as a therapeutic target in IMR-32 cells by testing a novel peptide CCT inhibitor, CT20p. Treatment with CT20p induced cell death in these neuroblastoma cells. The use of CCT2 as a biological indicator for detection of neuroblastoma cells shed in blood was examined by spiking IMR-32 cells into human blood and using an anti-CCT2 antibody for the identification of spiked cancer cells with the CellSearch system. Results showed that using CCT2 for the detection of neuroblastoma cells in blood was more effective than the conventional approach of using epithelial markers like cytokeratins. CCT2 plays an essential role in promoting the invasive capacity of neuroblastoma cells and thus offers the potential to act as a molecular target in the development of novel therapeutics and diagnostics for pediatric cancers.

GLI1 is a transcriptional effector at the terminal end of the Hedgehog signaling (Hh) pathway and is tightly regulated during embryonic development and tissue patterning/differentiation. GLI1 has low-level expression in differentiated tissues, however, in certain cancers, aberrant activation of GLI1 has been linked to the promotion of numerous hallmarks of cancer, such as proliferation, survival, angiogenesis, metastasis, metabolic rewiring, and chemotherapeutic resistance. All of these are driven, in part, by GLI1’s role in regulating cell cycle, DNA replication and DNA damage repair processes. The consequences of GLI1 oncogenic activity, specifically the activity surrounding DNA damage repair proteins, such as NBS1, and cell cycle proteins, such as CDK1, can be linked to tumorigenesis and chemoresistance. Therefore, understanding the underlying mechanisms driving GLI1 dysregulation can provide prognostic and diagnostic biomarkers to identify a patient population that would derive therapeutic benefit from either direct inhibition of GLI1 or targeted therapy towards proteins downstream of GLI1 regulation.

e15056 Background: The alpha fetoprotein (AFP) receptor is an oncofetal antigen and a novel target for cancer therapeutics. It is highly expressed on the surfaces of many cancers and myeloid derived suppressor cells (MDSCs) but absent on normal tissues. By conjugating a novel maytansinoid toxin to a recombinant form of human AFP (ACT-101), we can selectively deliver the toxin to cancer and MDSC cells while sparing normal cells, thereby enabling a combination of targeted and immune activating approaches against the tumor. Methods: Four ACT-101-maytansinoid conjugates with different protein-toxin ratios and different linker chemistries were initially investigated in a mouse COLO-205 xenograft model, resulting in the selection of ACT-903 as the lead candidate for further development. A second study performed in the same tumor model compared the effects of single intravenous doses of ACT-903 (10-50 mg/kg) to control groups receiving either vehicle or the unconjugated protein, ACT-101 (25 mg/kg). Tumor growth, survival and clinical observations were assessed for 60 days post tumor implantation. Both serum ACT-101 and maytansinoid levels were measured following the single dose. Results: In the first study, a statistically significant reduction in tumor volume was seen for all four conjugates compared to vehicle control (p < 0.05). For ACT-903, tumors continued shrinking even after treatment completion, becoming undetectable in 9 of 10 animals. All 10 mice in this group survived through Day 60 with no obvious signs of toxicity. In contrast, there were no survivors in the control group. In the single dose study of ACT-903, a significant reduction of tumor burden compared to vehicle was achieved by Day 14 (p < 0.05) in both the 40 and 50 mg/kg dose groups. Mice in these 2 groups received a second dose 15 days after the first dose, based on observed tumor re-growth. Survival was significantly prolonged in the 50 mg/kg (p = 0.0037) and 40 mg/kg group (p < 0.0001) with 7 of 10 in the 50 mg/kg group and 9 of 10 animals in the 40 mg/kg group surviving to Day 60. Similar doses of ACT-101 and ACT-903 produced comparable ACT-101 serum levels, suggesting that the pharmacokinetic profile of the conjugate is driven primarily by the protein component. Free maytansinoid levels at 4 hours were less than 0.01% of the injected dose of the conjugate, indicating that the conjugate is stable in circulation. Conclusions: The efficacy and tolerability of ACT-903 in an animal tumor model supports advancing this conjugate toward clinical use with a regimen of either once a week or once every other week administration.

TPS7077 Background: Patients with relapsed and/or refractory (r/r) myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML) who are not candidates for stem-cell transplantation have dismal outcomes. Novel therapies are needed. The nucleoside analogue NTX-301 (5-aza-4’-thio-2’-deoxycytidine or Aza-TdC) is incorporated into DNA, where it engages the active site of DNA methyltransferase I (DNMT1), a maintenance methyltransferase that contributes to the hypermethylation and silencing of tumor suppressor genes. DNMT1 can become trapped in a covalent complex with DNA, thus depleting free enzyme and inhibiting the normal maintenance methylation of CpG sites, resulting in re-activation of tumor suppressor genes. Preclinical data suggest a correlation between NTX-301 activity in leukemia xenograft models and decreased levels of DNMT1. NTX-301 offers an improvement over traditional DNA methyltransferase inhibitors (azacitidine, decitabine) by virtue of a higher incorporation rate into DNA at lower levels of cytotoxicity. Methods: This is a phase 1, open-label, multi-center, dose-escalation study to assess the safety, tolerability, and recommended myeloid monotherapy dose of NTX-301 in patients with r/r myeloid neoplasms. Patients ≥ 18 years with r/r higher-risk MDS (intermediate, high, very-high risk by IPSS-R), high-risk CMML (int-2/high by CPSS or CPSS-mol), or AML (marrow blasts ≤ 30% or WBC < 20,000 cells/µL without leukoreduction) are eligible. Other key eligibility criteria: Eastern Cooperative Oncology Group performance status 0–2; adequate cardiac, renal, and liver function; and resolved acute effects of any prior therapy. Successive cohorts of patients will receive escalating doses of NTX-301 starting from 2 mg QD. Each cycle is 21 days in duration (treatment for 5 days/week x 2 weeks and 1 week off). The trial incorporates accelerated titration design for dose level 1 and 2 followed by traditional 3+3 dose escalation design (3-6 patients per cohort) from dose level 3. Intrapatient dose escalation is allowed. NTX-301 treatment will continue until disease progression or unacceptable toxicity, whichever occurs first. Primary endpoints: incidence and severity of adverse events, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), biologically effective dose (BED). Secondary endpoints: PK parameters of NTX-301, PD assessment through global hypomethylation assay and other markers in blood and marrow, and clinical efficacy [overall response rate (ORR), complete remission (CR), marrow CR (mCR), partial remission (PR), stable disease (SD), hematologic improvement (HI) per mIWG criteria]. After the MTD/BED is identified, the safety and efficacy of NTX-301 will be explored further in selected myeloid malignancies. The study began enrolling patients in January 2021 and is still recruiting. Clinical trial information: NCT04167917.

Both the DNA damage response (DDR) and the mitotic checkpoint are critical for the maintenance of genomic stability. Among proteins involved in these processes, the ataxia-telangiectasia mutated (ATM) kinase is required for both activation of the DDR and the spindle assembly checkpoint (SAC). In mitosis without DNA damage, the enzymatic activity of ATM is enhanced; however, substrates of ATM in mitosis are unknown. Using stable isotope labeling of amino acids in cell culture mass spectrometry analysis, we identified a number of proteins that can potentially be phosphorylated by ATM during mitosis. This list is highly enriched in proteins involved in cell cycle regulation and the DDR. Among them, we further validated that ATM phosphorylated budding uninhibited by benzimidazoles 3 (Bub3), a major component of the SAC, on serine 135 (Ser135) both in vitro and in vivo. During mitosis, this phosphorylation promoted activation of another SAC component, benzimidazoles 1. Mutation of Bub3 Ser135 to alanine led to a defect in SAC activation. Furthermore, we found that ATM-mediated phosphorylation of Bub3 on Ser135 was also induced by ionizing radiation-induced DNA damage. However, this event resulted in independent signaling involving interaction with the Ku70-Ku80-DNA-PKcs sensor/kinase complex, leading to efficient nonhomologous end-joining repair. Taken together, we highlight the functional significance of the crosstalk between the kinetochore-oriented signal and double-strand break repair pathways via ATM phosphorylation of Bub3 on Ser135.

Resistance to radiation and chemotherapy in colorectal cancer (CRC) patients contribute significantly to refractory disease and disease progression. Herein, we provide mechanistic rationale for acquired or inherent chemotherapeutic resistance to the anti-tumor effects of 5-fluorouracil (5-FU) that is linked to oncogenic GLI1 transcription activity and NBS1 overexpression. Patients with high levels of GLI1 also expressed high levels of NBS1. Non-canonical activation of GLI1 is driven through oncogenic pathways in CRC, like the BRAFV600E mutation. GLI1 was identified as a novel regulator of NBS1 and discovered that by knocking down GLI1 levels in vitro, diminished NBS1 expression, increased DNA damage/apoptosis, and re-sensitization of 5-FU resistant cancer to treatment was observed. Furthermore, a novel GLI1 inhibitor, SRI-38832, which exhibited pharmacokinetic properties suitable for in vivo testing, was identified. GLI1 inhibition in a murine BRAFV600E variant xenograft model of CRC resulted in the same down-regulation of NBS1 observed in vitro as well as significant reduction of tumor growth/burden. GLI1 inhibition could therefore be a therapeutic option for 5-FU resistant and BRAFV600E variant CRC patients.

Anaplastic thyroid cancer (ATC) is an aggressive cancer with poor clinical prognosis. However, mechanisms driving ATC aggressiveness is not well known. Components of the DNA damage response (DDR) are frequently found mutated or aberrantly expressed in ATC. The goal of this study is to establish the functional link between histone acetyltransferase lysine (K) acetyltransferase 5 (KAT5, a critical DDR protein) and ATC invasiveness using clinical, in vitro and in vivo models. We analyzed the expression of KAT5 by immunohistochemistry and assessed its relationship with metastasis and overall survival in 82 ATC patients. Using cellular models, we established functional connection of KAT5 expression and C-MYC stabilization. We then studied the impact of genetically modified KAT5 expression on ATC metastasis in nude mice. In clinical samples, there is a strong correlation of KAT5 expression with ATC metastasis (P = 0.0009) and overall survival (P = 0.0017). At the cellular level, upregulation of KAT5 significantly promotes thyroid cancer cell proliferation and invasion. We also find that KAT5 enhances the C-MYC protein level by inhibiting ubiquitin-mediated degradation. Further evidence reveals that KAT5 acetylates and stabilizes C-MYC. Finally, we prove that altered KAT5 expression influences ATC lung metastases in vivo. KAT5 promotes ATC invasion and metastases through stabilization of C-MYC, demonstrating it as a new biomarker and therapeutic target for ATC.

Host genotype influences the severity of murine Lyme borreliosis, caused by the spirochetal bacterium Borrelia burgdorferi . C57BL/6 (B6) mice develop mild Lyme arthritis, whereas C3H/HeN (C3H) mice develop severe Lyme arthritis. Differential expression of interleukin 10 (IL-10) has long been associated with mouse strain differences in Lyme pathogenesis; however, the underlying mechanism(s) of this genotype-specific IL-10 regulation remained elusive. Herein we reveal a cAMP-mediated mechanism of IL-10 regulation in B6 macrophages that is substantially diminished in C3H macrophages. Under cAMP and CD14-p38 mitogen-activated protein kinase (MAPK) signaling, B6 macrophages stimulated with B. burgdorferi produce increased amounts of IL-10 and decreased levels of arthritogenic cytokines, including tumor necrosis factor (TNF). cAMP relaxes chromatin, while p38 increases binding of the transcription factors signal transducer and activator of transcription 3 (STAT3) and specific protein 1 (SP1) to the IL-10 promoter, leading to increased IL-10 production in B6 bone marrow-derived monocytes (BMDMs). Conversely, macrophages derived from arthritis-susceptible C3H mice possess significantly less endogenous cAMP, produce less IL-10, and thus are ill equipped to mitigate the damaging consequences of B. burgdorferi -induced TNF. Intriguingly, an altered balance between anti-inflammatory and proinflammatory cytokines and CD14-dependent regulatory mechanisms also is operative in primary human peripheral blood-derived monocytes, providing potential insight into the clinical spectrum of human Lyme disease. In line with this notion, we have demonstrated that cAMP-enhancing drugs increase IL-10 production in myeloid cells, thus curtailing inflammation associated with murine Lyme borreliosis. Discovery of novel treatments or repurposing of FDA-approved cAMP-modulating medications may be a promising avenue for treatment of patients with adverse clinical outcomes, including certain post-Lyme complications, in whom dysregulated immune responses may play a role.

MicroRNA, a class of small non-coding RNAs, play critical roles in the cellular response to DNA damage induced by ionizing irradiation (IR). Growing evidence shows alteration of miRNAs, in response to radiation, controls cellular radiosensitivity in DNA damage response pathways. However, it is less clear about the clinical relevance of miRNA regulation in radiosensitivity. Using an in vitro system, we conducted microarray to identify a miRNA signature to assess radiosensitivity. The data were validated by analyzing available Head and Neck Squamous Cell Carcinoma (HNSCC) samples in the cancer genome atlas (TCGA) database. A total of 27 miRNAs showed differential alteration in response to IR in an Ataxia-Telangiectasia Mutated (ATM) kinase-dependent manner. We validated the list and identified a five miRNA signature that can predict radiation responsiveness in HNSCC. Furthermore, we found that the expression level of ATM in these patients was correlated with the radiation responsiveness. Together, we demonstrate the feasibility of using a miRNA signature to predict the clinical responsiveness of HNSCC radiotherapy.

We report here the rational design and validation of a peptide inhibitor to the PD-1/PD-L1 interaction as an attempt to develop a viable alternative to current inhibitory antibodies. We demonstrated, by biolayer interferometry and in silico docking simulations, that a PD-L1 peptide mimetic (PL120131) can interfere with the PD-1/PD-L1 interaction by binding to PD-1. We show that PL120131 is capable of inhibiting PD-1 mediated apoptotic signaling pathway and rescuing Jurkat cells and primary lymphocytes from apoptosis. Additionally, we show that PL120131 treatment allows for CTL anti-tumor activity. Furthermore, PL120131 can maintain co-culture survivability and activity of T Cells in a 3D co-culture model better than the anti-PD-1 blocking antibody. Together, the characterization of this PD-1/PD-L1 inhibiting peptide provides insight regarding the ability to inhibit PD-L1 binding while maintaining CTL viability and activity that can further the development of alternatives to antibody based immunotherapies.

There is an unmet clinical need to identify biomarkers for breast cancer neoadjuvant chemotherapy. Here, using miRNA TaqMan Low-Density Arrays (TLDA), we analyzed the miRNA expression profile in pre-treatment needle aspiration tumor samples from patients who received taxane-anthracycline-based neoadjuvant chemotherapy. Although, in an unsupervised hierarchical cluster analysis, the total miRNA expression profile could not generate a tree with clear distinction between pathologic complete response (pCR) and non-pCR classes, we found that elevated expression of miR-125b and miR-141 was associated with non-pCR. In vitro experiments indicated that inhibition of miR-125b and miR-141 expression reduced cellular survival in response to taxane-anthracycline treatment. Furthermore, co-transfection with miR-125b and miR-141 mimics increased resistance of MCF7 and BT549 cells to taxane-anthracycline induced cytotoxicity. Pathway analyses indicated that many of the target proteins of miR-125b are involved in apoptotic pathways and cell cycle control. Together, we provide evidence that elevated miR-125b and 141 expression predicts a poor clinical responsiveness of taxane-anthracycline-based neoadjuvant chemotherapy.

Resistance to radiation and chemotherapy in colorectal patients is considered one of the major contributors of refractory disease and disease progression. Dysregulation or aberrant activation of the SHH/GLI1 signaling pathway, common to both colorectal cancer and other cancer subtypes, influences the integrity of the DNA damage response (DDR) pathway, which may impact tumor response to chemo- and radiation therapies. The DDR pathway is initiated in part by the recognition of a DNA lesion by a sensor protein and the recruitment NBS1 to that lesion. Subsequent repair is initiated by the Mre11, Rad50, NBS1 (MRN) complex formation. In this study, we investigated the expression of GLI1 and NBS1 in tissues samples from 188 patients diagnosed with colorectal cancer by immunohistochemistry and analyzed the clinical significance and prognostic relevance of elevated GLI1-NBS1 co-expression. GLI1 expression was positively associated with the NBS1 expression, and high expression of both in the biopsied specimens significantly correlated with poor patient survival. The result of a series of biochemical experiments directly links GLI1 transcriptional activity to the DNA damage repair pathway. First, NBS1 mRNA levels and protein were found to be reduced upon treatment with GANT61, a GLI inhibitor, indicating that GLI activity was required for NBS1 transcription. Second, overexpression of NBS1 in HT29 cells rescued GANT61-induced cell death by measure of cleaved caspase-3, indicating a significant role of induced DNA damage in the mechanism of GANT61-induced cell death in oncogenic GLI1 cancers. Third, this was verified by ChIP analysis of GLI1 binding to the NBS1 promotor region containing a putative GLI binding sequence. The efficacy of GLI1 inhibition as therapeutic strategy was then tested in parallel with 5-Fluorouracil (5-FU), a component of the standard-of-care therapy FOLFOX. In vitro, colorectal cell lines with GLI1-driven NBS1 expression demonstrated strong 5-FU resistance. These cells were re-sensitized to 5-FU by silencing the expression of NBS1 with siRNA, or with combination therapy involving 5-FU and GANT61. This suggests that a first-in class chemotherapeutic strategy may provide additional treatment options for patients demonstrating 5-FU resistance during the course of their chemotherapy. While GANT61 is commonly used to inhibit GLI in vitro, it exhibits poor pharmacokinetic properties and is not a viable drug for the clinic. To that end, we present a novel GLI1 antagonist SR38832, which exhibits a more stable pharmacokinetic profile than GANT61 in xenograft mouse models and lower IC50 in colon cancer cell lines. In a human colorectal cancer xenograft mouse model, SR38832 significantly inhibited both tumor growth and proliferation. Not only has the identification of SR38832 provided an in vivo tool to study GLI oncogenic activity, but SR38832 is also structural lead to be optimized through an iterative medicinal chemistry approach. Citation Format: Ruowen Zhang, Justin Avery, Jinlu Ma, Theresa Nguyen, Mark J Suto, Bo Xu, Rebecca J Boohaker. Targeting GLI1-mediated NBS1 transcription overcomes 5-FU resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A103. doi:10.1158/1535-7163.TARG-19-A103

The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration.

Tumor cells exploit immune checkpoints by expressing checkpoint ligands such as PD-L1, effectively masking them from immune destruction. Programmed cell death- 1 (PD-1), plays a major role in tumor immune escape. The interaction of PD-1/PD-L1 inhibits cytotoxic T lymphocyte (CTL) proliferation, induces apoptosis of tumor-specific T cells, and promotes the resistance of tumor cells to CTL attack. Monoclonal antibodies to these proteins have been developed and approved for use in the clinic. Theoretically, peptide mimics, which have the benefit of lower half-life and significantly smaller size, are able to penetrate into solid tumors and tissues better than antibodies, and can function as inhibitors for the PD1/PD-L1 interaction. Here we report for the first time the rational design and validation of a peptide inhibitor to the PD-1/PD-L1 immune checkpoint as a viable alternative to current inhibitory antibodies. We demonstrated, by biolayer interferometry and in silico docking simulations, that a PD-L1 peptide mimetic (PL120131) can interfere with the PD-1/PD-L1 interaction by binding to PD-1. We established the binding constant for the interaction and PL120131 reduces association of PD-L1 in a dose dependent manner. We show that PL120131 is capable of inhibiting PD-1 mediated apoptotic signaling pathway and rescuing Jurkat cells and primary lymphocytes from apoptosis. Additionally, we show that PL120131 treatment allows for CTL anti-tumor activity. Furthermore, PL120131 can maintain co-culture survivability and activity of T Cells in a 3D co-culture model. Together, the characterization of this PD-1/PD-L1 inhibiting peptide provides insight regarding the ability to inhibit PD-L1 binding while maintaining CTL viability and activity. Understanding the mechanism(s) of action of this peptide has yielded information on the pathways that are drivers of immune evasion in a tumor microenvironment, which may improve and synergize with other forms of immunotherapy, thus increasing efficacy of cancer treatment. Outcomes from this study have provided a path forward to further development of small molecule inhibitors to the PD-1/PD-L1 interaction, and outlines a strategy for the design and development of small molecules to additional cell-surface checkpoint proteins. Citation Format: Rebecca J. Boohaker, Vijaya Sambandam, Mark Suto, Bo Xu. The rational design and evaluation of a peptide inhibitor of the PD-1/PD-L1 interaction [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 5668.

Apoptosis is a complex process essential for normal tissue development and cellular homeostasis. While biochemical events that occur late in the apoptotic process are better characterized, early physiological changes that initiate the progression of cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to growth factor withdrawal, experienced a rapid and transient rise in cytosolic pH. We found that the protein responsible was the pH-regulating, plasma membrane protein Na+/H+exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of the stress-activated kinase, p38 MAP kinase. In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a cell death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to an apoptotic stimulus, and expression of which protected cells from serum withdrawal- induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Analysis of serine phosphorylation showed that phosphorylation of NHE1 during apoptosis decreased upon mutation of Ser726 and Ser729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of a cell death response.

MicroRNAs are a critical class of regulators for cells to deal with DNA damage. Abnormal miRNA function is associated with tumor initiation and progression, and altered miRNA expression found in tumor tissues are frequently associated with heterogeneity of tumor responses to therapeutic agents, including radiotherapy. In this chapter, we review recent advances of the functional role of microRNAs in the context of the DNA damage response, tissue specific tumor initiation and progression. We further discuss clinical implications of using miRNA signatures as biomarkers for radiosensitivity and targeting specific miRNAs as therapeutic approaches.

The Fc receptor-like protein 5 (FCRL5) on B cells has both an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence and two consensus immunoreceptor tyrosine-based inhibitory motifs (ITIM) in its cytoplasmic region. To evaluate its signaling potential, we expressed constructs for chimeric molecules composed of the cytoplasmic region of FCRL5 and the extracellular and transmembrane regions of the IgG Fc receptor FcγRIIB in a B cell line lacking an endogenous Fc receptor. Coligation of this fusion protein with the B cell receptor (BCR) inhibited BCR-mediated calcium mobilization, intracellular tyrosine phosphorylation, and Erk kinase activation. Our mutational analysis indicated that, whereas tyrosines in both the inhibitory and activation motifs are phosphorylated after ligation, only those in ITIMs influence BCR-mediated signaling. This FCRL5 inhibitory effect was mediated through dual ITIM recruitment of the SH2-containing protein tyrosine phosphatase, SHP-1, which in turn dephosphorylates the ITAM-based tyrosines in BCR Igα/Igβ heterodimers. An FCRL5 inhibitory effect on BCR signaling was likewise demonstrable for primary B cells. Although its ligand is presently unknown, we conclude that FCRL5 has the functional potential to serve as an inhibitory coreceptor on mature B cells in humans.

Mitotic kinesin Eg5 is an attractive anti-cancer drug target. Discovery of Eg5 inhibitors has been focused on targeting the “monastrol-binding site”. However, acquired drug resistance has been reported for such inhibitors. Therefore, identifying new Eg5 inhibitors which function through a different mechanism(s) could complement current drug candidates and improve drug efficacy. In this study, we explored a novel allosteric site of Eg5 and identified new Eg5 inhibitors through structure-based virtual screening. Experiments with the Saturation-Transfer Difference (STD) NMR demonstrated that the identified Eg5 inhibitor SRI35566 binds directly to Eg5 without involving microtubules. Moreover, SRI35566 and its two analogs significantly induced monopolar spindle formation in colorectal cancer HCT116 cells and suppressed cancer cell viability and colony formation. Together, our findings reveal a new allosteric regulation mechanism of Eg5 and a novel drug targeting site for cancer therapy.

The Ataxia-Telangiectasia Mutated (ATM) Kinase, mutation of which is responsible for the autosomal recessive disorder Ataxia-Telangiectasia, is an essential element for cells to deal with DNA double-strand breaks. In the absence of DNA damage, it has also been recognized that ATM has a role in optimizing the spindle assembly checkpoint during mitosis. In the process, ATM is activated at a lower level (compared to the DNA damage response) which requires phosphorylation by the Aurora-B kinase. Known ATM targets in mitosis include kinetochore proteins Bub1 and Mad1. However, it has become clear that ATM-mediated phosphorylation in mitosis is distinct from that in the DNA damage response. Therefore, we performed an unbiased assay to identify ATM substrates in mitosis in order to study the functional significance of mitotic ATM activation. To achieve our goal, we utilized a stable isotope labeling by amino acid (SILAC) /Peptide IP with phosphor-specific SQ/TQ antibodies and mass spectrometry (MS). About 18 proteins were identified as substrates of mitotically activated ATM in the assay. We then conducted bioinformatic analyses for the identified proteins using PANTHER and Ingenuity programs. With many proteins having their known functions in mitosis, we found a much smaller number, but highly interconnected, pool of proteins in the ATM substrate list as compared to that of the DNA damage response. Among them, we validated several phosphorylation events using an in vitro kinase assay. Furthermore we present data to demonstrate the functional significance of ATM-mediated phosphorylation. Taken together, our data provide a comprehensive picture of how mitotically activated ATM signals to downstream targets to fine-tune the mitotic checkpoint. Citation Format: Rebecca J. Boohaker, Qinghua Zeng, Bo Xu. Identification and characterization of ATM substrates in mitosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 180.

The Speckle type Poz Protein (SPOP), an E3 ubiquitin ligase adaptor, has recently been identified as the gene that has the most common somatic point mutations in prostate cancer. SPOP mutations are associated with genomic alterations, indicating a role for SPOP in the maintenance of genome stability. We, and others, have recently demonstrated a critical role of SPOP in the DNA damage response (DDR), suggesting SPOP mutants may represent a subgroup of patients that have hyper sensitivity to DNA damaging therapies. However, how SPOP mutations might impact its function and their roles in the progress of prostate tumorigenesis remain to be extensively studied. Genomic studies have found that SPOP mutations are clustered within its substrate binding pocket. Of a particular interest, Serine119 is found to be frequently mutated to Asparagine (S119N). Using computational modeling, we found that Ser119 resides in the SBC-MATH binding interface and is in close contact with the non-polar residue of the SPOP-binding consensus motif. Therefore we hypothesized that phosphorylation of Serine 119 might directly affect substrate binding. First we characterized prostate cancer cells expressing this mutation and we found that they displayed a suboptimal DNA damage response with prolonged DNA repair. Using an in situ proximity ligation assay, we demonstrate that Serine 119 is essential for SPOP ionizing irradiation induced interaction with the Ataxia Telangiectasia Mutated kinase (ATM), a major hub protein in the DDR. With the evidence of SPOP being a phospho-protein, we further provide in vitro evidence that ATM phosphorylates SPOP on Serine 119 in response to DNA damage. Characterization of the functional significance of ATM-mediated SPOP phosphorylation indicates a wide range of downstream targets regulating cell cycle progression and DNA repair. Taken together, our data reveal a critical pathway linking ATM and SPOP in regulation of prostate cancer initiation and therapeutic responses to DNA damage. This also provides the first evidence of a pathophysiological relevant mutation linked to ATM phosphorylation in the DDR. Citation Format: Joshua Fried, Rebecca Boohaker, Qinghua Zeng, Wei Zhang, Bo Xu. Characterization of the ATM-SPOP pathway in prostate cancer tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2752.

Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

The BCL2 family of proteins tightly regulates the delicate balance between life and death. Bax, a proapoptotic member of this family, acts as the penultimate factor in the apoptotic cascade by releasing apoptogenic factors such as Cytochrome C from the mitochondrial lumen. The mechanism of mitochrondrial permeabilization by BAXis not well defined. What is known is that BAX translocates to and aggregates at the outer mitochondrial membrane before cytochrome C is released, implying the insertion of the protein occurs after the aggregation event. In this work, we have evaluated the function of the oligomerization state of BAX on the insertion of the protein into artificial membranes.

To cope with constant challenges toward the fidelity of genome by DNA damaging insults (both endogenous and exogenous), activation of the DNA damage response (DDR) is an essential step in preventing genomic instability. Studies of mammalian mechanisms have revealed that there are comprehensive, coordinated pathways that, when activated, serve to regulate the DDR. Recently, we have shown that hyperactive DDR mediated by the Ataxia-Telangiectasia Mutated (ATM) kinase is linked to activation of the Zinc finger protein SNAI1 (Snail). Activated Snail in metastasis is well-characterized by its role as transcriptional repressor of E-cadherin thereby inducing an epithelial-to-mesenchymal transition (EMT). Here we report that, in response to DNA damage, activation of the ATM-Snail pathway, mediated by Snail Serine 100 phosphorylation, also contributes to the regulation of the cellular sensitivity to ionizing radiation. Expression of a hyperactive Snail led to radioresistance. More interestingly, the Serine 100 to Glutamic Acid mutant variant of Snail displayed an enhanced invasion index after low dose radiation, independent of survival. Functional genomics and proteomics studies revealed downstream targets and interacting partners of Snail in the DDR. Together, we highlight the functional significance of the ATM-Snail pathway in the DDR. Citation Format: Rebecca J. Boohaker, Joshua Fried, Xiaoli Cui, Bo Xu. Delineation of the ATM-Snail pathway in the DNA damage response. [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 3939. doi:10.1158/1538-7445.AM2014-3939

Ataxia-telangiectasia mutated (ATM) kinase, the mutation of which causes the autosomal recessive disease ataxia-telangiectasia, plays an essential role in the maintenance of genome stability. Extensive studies have revealed that activated ATM signals to a massive list of proteins to facilitate cell cycle checkpoints, DNA repair, and many other aspects of physiological responses in the event of DNA double-strand breaks. ATM also plays functional roles beyond the well-characterized DNA damage response (DDR). In this review article, we discuss the recent findings on the molecular mechanisms of ATM in DDR, the mitotic spindle checkpoint, as well as hyperactive ATM signaling in cancer invasion and metastasis.

BACKGROUND: The marked difference in metabolism observed between tumor and normal cells could contribute to the development of invasive and metastatic forms of breast cancer. The problem is that while patients diagnosed with invasive forms of breast cancer may be initially responsive to treatment, a significant number develop relapsing and even metastatic disease. There is a critical unmet need to develop new therapeutic approaches for patients diagnosed with invasive forms of breast cancer that are effective given the unique metabolism of tumor cells. METHODS: We examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminus of Bax. For delivery to cells, the amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs). NPs were made using aliphatic hyperbranched polyester (HBPE) that incorporated surface carboxylic groups and interior hydrophobic cavities for encapsulation of CT20p. To examine the cytotoxic potential and targeting capacity of CT20p-HBPE-NPs, we treated MDA-MB-231 breast cancer cells and MCF-10A breast epithelial cells with the peptide-nanoparticle combination and measured changes in mitochondrial function, cell metabolism and induction of apoptotic and non-apoptotic cell death. The ability of CT20p-NP-HBPE to cause tumor regression was examined by subcutaneously implanting MDA-MB-231 cells in nude mice. RESULTS: Initial studies showed that CT20p caused the release of calcein from mitochondrial-like lipid vesicles, without disrupting vesicle integrity, and, when expressed as a fusion protein in cells, localized to mitochondria. While the peptide alone had little effect upon intact cells, likely not penetrating the plasma membrane, when encapsulated and delivered by nanoparticles, CT20p-HBPE-NPs proved an effective killer of breast cancer cells. CT20p-HBPE-NPs initiated non-apoptotic cell death within 3 hours of treatment by targeting mitochondria and deregulating cellular metabolism. Nanoparticles alone or nanoparticles encapsulating a control peptide had minimal effects. The cytotoxicity of CT20p-HPBE-NPs was most pronounced in breast cancer cells, sparing normal, epithelial cells. In implanted breast tumors, CT20p-HBPE-NPs accumulated in tumors within 24 hours and reduced tumor burden by 50-80%. CONCLUSION: These results reveal the innovative features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies that target the unique metabolism of cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-13.

The success of stem cell transplantation depends on the reconstitution of the recipient’s immune system. The lymphocyte growth factor, Interleukin-7 (IL-7), has the potential to improve immune reconstitution; however, at the supraphysiologic doses being tested in clinical trials, consequences such as down-regulation of the IL-7 receptor (IL-7R), altered CD4:CD8 ratios or a change in the homing of T-cells could affect the quality of the immune reconstitution. Ourresearch objective was to design a new form of IL-7 that retained its capacity for supporting lymphocyte expansion without the need for a high dosing regimen. To this end, we developed IL-7tv, an IL-7R ligand with a mutation adjacent to the receptor binding domain. Using surface plasmon resonance, (SPR), we showed that, compared to IL-7, binding of IL-7tv to IL-7Ra was slightly reduced, while the total binding constant (KD) remained the same. This could be due to the mutation decreasing accessibility of IL-7tv to the IL-7Ra. To test whether IL-7tv would deliver a sustained growth signal through the IL-7R, we used an IL-7-dependent murine T-cell, line, D1, human T-cells, and hematopoietic stem cells (HSCs). Using D1 cells, we found that IL-7tv supported survival through phosph-STAT5 activation, at doses 10-fold lower than the optimal concentration of native IL-7, and did not down-regulate the IL-7R. Applied to human T-cells in culture, IL-7tv promoted the growth of both CD4 and CD8 subsets at a dose 40-fold less than IL-7, without causing expression of the activation receptor, CD69. Importantly, IL-7tv, at lower doses, drove the ex vivo expansion of CD34+ HSCs more effectively than IL-7. These results support the use of IL-7tv as a novel agent for immune reconstitution and indicate that modulation of ligand binding to the IL-7R is a powerful means by which to harness the growth potential of the cytokine.

3085 Background: Patients with metastatic breast cancer may be initially responsive to treatment, but a significant number develop refractory disease. There is a critical unmet need to develop effective therapeutic approaches given the unique metabolism of tumor cells. Methods: We examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminus of Bax. For delivery to cells, the amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs). NPs were made using aliphatic hyperbranched polyester (HBPE) that incorporated surface carboxylic groups and interior hydrophobic cavities for encapsulation of CT20p. To examine the cytotoxic potential and targeting capacity of CT20p-NP-HBPE, we treated MDA-MB-231 and MCF-10A breast cancer cell lines with the combination and measured changes in mitochondrial function, cell metabolism and induction of cell death. The ability of CT20p-NP-HBPE to cause tumor regression was examined by subcutaneously implanting MDA-MB-231 cells in nude mice. Results: Initial results showed that CT20p caused the release of calcein from mitochondrial-like lipid vesicles, without disrupting vesicle integrity, and, when expressed as a fusion protein in cells, localized to mitochondria. While the peptide alone had little effect upon intact cells, when encapsulated and delivered by nanoparticles, CT20p-HBPE-NPs proved an effective killer of breast cancer cells. CT20p-NP-HBPE initiated non-apoptotic cell death within 3 hours of treatment by targeting mitochondria and deregulating cellular metabolism. Nanoparticles alone or nanoparticles encapsulating a control peptide had minimal effects. The cytotoxicity of CT20p-NP-HPBE was most pronounced in breast cancer cells, sparing normal, epithelial cells. In implanted breast tumors, CT20p-NP-HBPE accumulated in tumors within 24 hours and reduced tumor burden by 50-80%. Conclusions: These results reveal the innovative features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies that target the unique metabolism of cancer cells.

The Bcl-2 protein family modulates the balance between cellular life and death. Bax, a pro-apoptotic member of this family, participates in mitochondria-mediated apoptosis but also supports mitochondrial bioenergetics. In previous work, we showed that a peptide derived from the alpha 9-helix of Bax is cytotoxic, specifically when used to treat breast cancer cells. Such structural and biochemical studies are useful for identifying functional domains within Bax that can be developed into therapeutic peptides, but are limiting when ascribing function to domains at a molecular level. Computational modeling and molecular dynamics simulations of Bax provides a high-power analysis of the subtle changes that modulate the regulatory and effector activities of the protein. This study seeks to elucidate the consequence of alterations in micro-domains that confer activity, apoptotic or otherwise, to the protein. Molecular dynamics simulation was used to determine how the trans-membrane region of Bax conferred stability to the protein, identifying regions of high flexibility and differential exposure. Computationally rotating the Ψ angle at Pro168 altered the position of the α9-helix of Bax, mimicking the “active” and “inactive” forms of Bax. We found that “active” Bax, but not “inactive” Bax, presented a more relaxed structure which altered the conformation and exposure of the BH3 domain, potentially enhancing interactions among the family of Bcl-2 proteins. Since dysregulation of Bcl-2 proteins is implicated in a number of cancers, examining the basic structural dynamics at a molecular level yielded information that can aid in the design novel Bcl-2 family-targeting chemotherapeutics. Citation Format: Annette Khaled, Rebecca Boohaker, Craig Finch, Sergio Tafur. Structural insights into the dynamic functionality of Bax through molecular dynamics simulations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5138. doi:10.1158/1538-7445.AM2013-5138

The Bcl-2 family proteins maintain the balance of life and death in a cell. Bax, a pro-apoptotic member, translocates to the mitochondrial membrane in response to diverse death stimuli, initiating a cascade of programmed events that lead to cell death. Tumor progression involves deregulation of this process, in part, through mutations that cause aberrant expression of anti-apoptotic members of the Bcl-2 family that normally sequester and inhibit Bax. This study seeks to identify functional domains of Bax that can be developed into therapeutic peptides to promote the death of cancer cells resistant to existing chemotherapeutics. Our goal was to generate a peptide, much like antimicrobial peptides, that could target and disrupt mitochondrial membranes. To this end, we discovered that the C-terminal, alpha-9 helix of Bax (CT20), when tagged to EGFP or a destabilization domain (DD), could bind mitochondria and cause cell death, mimicking the activity of full-length Bax. Cell death was measured by morphological changes, such as membrane blebbing, as well as DNA fragmentation. Targeted mutagenesis of two adjacent lysines at the carboxyl end of the CT20 peptide demonstrated that these residues enabled mitochondrial membrane association. Expression of the CT20 peptide in the presence or absence of endogenous Bax resulted in cytotoxicity, indicating that the death mechanism engaged could function independently of the Bcl-2 family. These findings suggest the CT20 peptide has the potential to be developed into a viable therapeutic agent in the treatment of a broad range of cancer types. 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 2010. doi:1538-7445.AM2012-2010

The dual functionality of the tumor suppressor, BAX, is implied by the non-apoptotic functions of other pro-apoptotic BCL-2 family proteins. To explore this, the mitochondrial morphology and ATP producing capability in both BAX-containing and BAX-deficient HCT-116 colon cancer cells were examined. While BAX deficient cells maintain the same mitochondrial mass as their BAX containing counterparts, these mitochondria were visibly smaller, more compact, and unable to maintain an organized network. Oxygen consumption and ATP levels were also reduced in these cells, a phenomenon that was replicated upon treatment of BAX-containing cells with the uncoupling agent, FCCP. These results, coupled with a marked decrease in citrate synthase activity, suggested that cells lacking BAX have a deficiency in the ability to generate ATP through oxidative phosphorylation. Inhibition of endogenous BAX through treatment with small interfering RNAs reinforced the necessity of BAX activity in the maintenance of aerobic respiration and the production of ATP. An examination of the localization of full length and C-terminal truncated BAX in non-apoptotic, BAX deficient cells, indicated that a small fraction of the existing cytosolic BAX is needed at the mitochondrial membrane to maintain ATP production. However, this function is not dependent upon the C-terminal helix. Co-expression of BAX and BCL-2 resulted in a subsequent loss of ATP production, implying that, even under non-apoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX associated with the mitochondria is necessary to maintain mitochondrial bioenergetics. The discovery of this novel, non-apoptotic role for BAX in the maintenance of ATP synthesis and suggests that complete abrogation of BAX would be detrimental to cancer cells dependent on energy production via aerobic respiration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3794. doi:10.1158/1538-7445.AM2011-3794