Targeted therapy of multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy and the second most common hematologic cancer. MM is characterized by the accumulation of malignant plasma cells within the bone marrow, and presents clinically with a broad range of symptoms, including hypercalcemia, renal insufficiency, anemia, and lytic bone lesions. MM is a heterogeneous disease associated with genomic instability, where patients may express multiple genetic abnormalities that affect several oncogenic pathways. Commonly detected genetic aberrations are translocations involving immunoglobulin heavy chain (IgH) switch regions (chromosome 14q32) and oncogenes such as c-maf [t(14:16)], cyclin D1 [t(11:14)], and FGFR3/MMSET [t(4:14)]. Advances in the basic understanding of MM and the development of novel agents, such as the immunomodulatory drugs (IMiDs) thalidomide and lenalidomide and the proteasome inhibitor bortezomib, have increased therapeutic response rates and prolonged patient survival. Despite these advances MM remains incurable in the majority of patients, and it is therefore critical to identify additional therapeutic strategies and targets for its treatment. In this chapter, we review the underlying genetic components of MM and discuss the results of recent clinical trials that demonstrate the effectiveness of targeted agents in the management of MM. In addition, we discuss experimental therapies that are currently in clinical development along with their molecular rationale in the treatment of MM.

Stimulation of natural killer cells with small molecule inhibitors of CD38 for the treatment of neuroblastoma.

High-risk neuroblastoma (NB) accounts for 15% of all pediatric cancer deaths. Refractory disease for high-risk NB patients is attributed to chemotherapy resistance and immunotherapy failure. The poor prognosis for high-risk NB patients demonstrates an unmet medical need for the development of new, more efficacious therapeutics. CD38 is an immunomodulating protein that is expressed constitutively on natural killer (NK) cells and other immune cells in the tumor microenvironment (TME). Furthermore, CD38 over expression is implicated in propagating an immunosuppressive milieu within the TME. Through virtual and physical screening, we have identified drug-like small molecule inhibitors of CD38 with low micromolar IC50 values. We have begun to explore structure activity relationships for CD38 inhibition through derivatization of our most effective hit molecule to develop a new compound with lead-like physicochemical properties and improved potency. We have demonstrated that our derivatized inhibitor, compound 2, elicits immunomodulatory effects in NK cells by increasing cell viability by 190 ± 36% in multiple donors and by significantly increasing interferon gamma. Additionally, we have illustrated that NK cells exhibited enhanced cytotoxicity toward NB cells (14% reduction of NB cells over 90 minutes) when given a combination treatment of our inhibitor and the immunocytokine ch14.18-IL2. Herein we describe the synthesis and biological evaluation of small molecule CD38 inhibitors and demonstrate their potential utility as a novel approach to NB immunotherapy. These compounds represent the first examples of small molecules that stimulate immune function for the treatment of cancer.

Secreted frizzled related-protein 2 is prognostic for human pancreatic cancer patient survival and is associated with fibrosis.

Pancreatic adenocarcinoma (PDAC) is one of the deadliest cancers, with five-year survival rates of 9%. We hypothesized that secreted frizzled-related protein 2 (SFRP2) may influence stromal growth in pancreatic cancer, since it increases fibrosis and collagen production in non-neoplastic pathologies. We assessed SFRP2 value as a biomarker and assessed its function in PDAC. SFRP2 gene expression in patients with PDAC was analyzed using TCGA data. Disease free survival (DFS) was analyzed using Kaplan Meier test. The effect of KRAS inhibition on SFRP2 expression in PDAC cells was assessed. The associations of stromal content with SFPR2 mRNA and protein with fibrosis were analyzed. The role of SFRP2 in mesenchymal transformation was assessed by western blot in fibroblasts. Of all cancers in TCGA, SFRP2 levels were highest in PDAC, and higher in PDAC than normal tissues (n= 234, p= 0.0003). High SFRP2 levels correlated with decreased DFS (p= 0.0097). KRAS inhibition reduced SFRP2 levels. Spearman correlation was 0.81 between stromal RNA and SFRP2 in human PDAC, and 0.75 between fibrosis and SFRP2 levels in PDAC tumors. SFRP2-treated fibroblasts displayed mesenchymal characteristics. SFRP2 is prognostic for PDAC survival, regulated by KRAS, and associated with PDAC fibrosis.

High-resolution imaging and antitumor effects of GFP(+) bone marrow-derived cells homing to syngeneic mouse colon tumors.

Bone marrow-derived cells (BMDCs) participate in the growth and spread of tumors of the breast, brain, lung, and stomach. To date, there are limited reports of bone marrow involvement in colon cancer pathogenesis, but such findings would have the potential to generate novel treatments for colon cancer patients. We have established a mouse model for imaging BMDCs from whole tumor to single-cell resolution, whereby the bone marrow of lethally irradiated host animals is reconstituted with EGFP-expressing bone marrow cells from matched TgActb(EGFP) donors. The BM transplants yield mice with fluorescently labeled bone marrow, and so BMDCs can subsequently be monitored within a tumor through optical imaging. Successful BM reconstitution was confirmed at 8 weeks after transplantation, when surviving BALB/c mice were injected with CT26 mouse colon cancer cells. We find that up to 45% of cells dissociated from the tumors are GFP(+) and approximately 50% of Lin(+), CD11b(+), and CD3(+) cells express high levels of GFP. Notably, tumor growth is reduced in BM transplanted animals, compared with untransplanted host mice or EGFP-expressing BM donor mice. A needed next step is to separate the molecular and cellular (eg, T cells, NK cells, macrophages) bases of the antitumor effect of the BMDCs from any protumorigenic effect that could be subverted for therapeutic gain.

Effect of time to infusion of autologous stem cells (24 vs. 48 h) after high-dose melphalan in patients with multiple myeloma.

High-dose melphalan (HD-Mel) is considered the current standard of care among the preparative regimens used in autologous peripheral blood stem cell transplantation (SCT) for multiple myeloma (MM), but optimal time and schedule of administration is not defined. We retrospectively analyzed outcomes and toxicities of HD-Mel administered on day -2 vs. day -1 before autologous stem cells infusion. A total of 138 consecutive MM patients treated at Penn State Hershey Cancer Institute between 2007 and 2010 were included in this study. No difference in time to hematopoietic recovery, common SCT-related toxicities, and clinical outcomes was seen between patients who received HD-Mel on day -2 (group A, n = 47), and those who received it on day -1 (group B, n = 91). Prompt and full hematopoietic recovery occurred even when stem cells were infused between 8 and 24 h after completion of chemotherapy. In the absence of prospective and randomized data, we conclude that a single I.V. infusion of HD-Mel on day -1 is a safe and effective practice, and the so-called 'day of rest' before the transplant appears not to be necessary.

PDI inhibitor LTI6426 enhances panobinostat efficacy in preclinical models of multiple myeloma.

The histone deacetylase inhibitor (HDACi), panobinostat (Pano), is approved by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) for treatment of relapsed/refractory multiple myeloma (MM). Despite regulatory approvals, Pano is used on a limited basis in MM due largely to an unfavorable toxicity profile. The MM treatment landscape continues to evolve, and for Pano to maintain a place in that paradigm it will be necessary to identify treatment regimens that optimize its effectiveness, particularly those that permit dose reductions to eliminate unwanted toxicity. Here, we propose such a regimen by combining Pano with LTI6426, a first-in-class orally bioavailable protein disulfide isomerase (PDI) inhibitor. We show that LTI6426 dramatically enhances the anti-MM activity of Pano in vitro and in vivo using a proteasome inhibitor resistant mouse model of MM and a low dose of Pano that exhibited no signs of toxicity. We go on to characterize a transcriptional program that is induced by the LTI6426/Pano combination, demonstrating a convergence of the two drugs on endoplasmic reticulum (ER) stress pathway effectors ATF3 (Activating Transcription Factor 3), DDIT3/CHOP (DNA Damage Inducible Transcript 3, a.k.a. C/EBP Homologous Protein), and DNAJB1 (DnaJ homolog subfamily B member 1, a.k.a. HSP40). We conclude that LTI6426 may safely enhance low-dose Pano regimens and that ATF3, DDIT3/CHOP, and DNAJB1 are candidate pharmacodynamic biomarkers of response to this novel treatment regimen.

Selective targeting of CD38 hydrolase and cyclase activity as an approach to immunostimulation.

The ectoenzyme CD38 is highly expressed on the surface of mature immune cells, where they are a marker for cell activation, and also on the surface of multiple tumor cells such as multiple myeloma (MM). CD38-targeted monoclonal antibodies (MABs) such as daratumumab and isatuximab bind to CD38 and promote cancer cell death by stimulating the antitumor immune response. Although MABs are achieving unprecedented success in a percentage of cases, high rates of resistance limit their efficacy. Formation of the immunosuppressive intermediate adenosine is a major route by which this resistance is mediated. Thus there is an urgent need for small molecule agents that boost the immune response in T-cells. Importantly, CD38 is a dual-function enzyme, serving as a hydrolase and a nicotinamide adenine dinucleotide (NAD+) cyclase, and both of these activities promote immunosuppression. We have employed virtual and physical screening to identify novel compounds that are selective for either the hydrolase or the cyclase activity of CD38, and have demonstrated that these compounds activate T cells in vitro. We are currently optimizing these inhibitors for use in immunotherapy. These small molecule inhibitors of the CD38-hydrolase or cyclase activity can serve as chemical probes to determine the mechanism by which CD38 promotes resistance to MAB therapy, and could become novel and effective therapeutic agents that produce immunostimulatory effects. Our studies have identified the first small molecule inhibitors of CD38 specifically for use as immunostimulants.

Syrbactin proteasome inhibitor TIR-199 overcomes bortezomib chemoresistance and inhibits multiple myeloma tumor growth in vivo.

Multiple myeloma (MM) and mantle cell lymphoma (MCL) are blood cancers that respond to proteasome inhibitors. Three FDA-approved drugs that block the proteasome are currently on the market, bortezomib, carfilzomib, and ixazomib. While these proteasome inhibitors have demonstrated clinical efficacy against refractory and relapsed MM and MCL, they are also associated with considerable adverse effects including peripheral neuropathy and cardiotoxicity, and tumor cells often acquire drug resistance. TIR-199 belongs to the syrbactin class, which constitutes a novel family of irreversible proteasome inhibitors. In this study, we compare TIR-199 head-to-head with three FDA-approved proteasome inhibitors. We demonstrate that TIR-199 selectively inhibits to varying degrees the sub-catalytic proteasomal activities (C-L/ß1, T-L/ß2, and CT-L/ß5) in three actively dividing MM cell lines, with Ki50 (CT-L/ß5) values of 14.61 ±â€¯2.68 nM (ARD), 54.59 ±â€¯10.4 nM (U266), and 26.8 ±â€¯5.2 nM (MM.1R). In most instances, this range was comparable with the activity of ixazomib. However, TIR-199 was more effective than bortezomib, carfilzomib, and ixazomib in killing bortezomib-resistant MM and MCL cell lines, as judged by a low resistance index (RI) between 1.7 and 2.2, which implies that TIR-199 indiscriminately inhibits both bortezomib-sensitive and bortezomib-resistant MM and MCL cells at similar concentrations. Importantly, TIR-199 reduced the tumor burden in a MM mouse model (p < 0.01) confirming its potency in vivo. Given the fact that there is still no cure for MM, the further development of TIR-199 or similar molecules that belong to the syrbactin class of proteasome inhibitors is warranted.

Design of Hydrazide-Bearing HDACIs Based on Panobinostat and Their p53 and FLT3-ITD Dependency in Antileukemia Activity.

Here, we present a new series of hydrazide-bearing class I selective HDAC inhibitors designed based on panobinostat. The cap, linker, and zinc-binding group were derivatized to improve HDAC affinity and antileukemia efficacy. Lead inhibitor 13a shows picomolar or low nanomolar IC50 values against HDAC1 and HDAC3 and exhibits differential toxicity profiles toward multiple cancer cells with different FLT3 and p53 statuses. 13a indirectly inhibits the FLT3 signaling pathway and down-regulates master antiapoptotic proteins, resulting in the activation of pro-caspase3 in wt-p53 FLT3-ITD MV4-11 cells. While in the wt-FLT3 and p53-null cells, 13a is incapable of causing apoptosis at a therapeutic concentration. The MDM2 antagonist and the proteasome inhibitor promote 13a-triggered apoptosis by preventing p53 degradation. Furthermore, we demonstrate that apoptosis rather than autophagy is the key contributing factor for 13a-triggered cell death. When compared to panobinostat, 13a is not mutagenic and displays superior in vivo bioavailability and a higher AUC0-inf value.

ATF3 Coordinates Antitumor Synergy between Epigenetic Drugs and Protein Disulfide Isomerase Inhibitors.

Histone deacetylase inhibitors (HDACi) are largely ineffective in the treatment of solid tumors. In this study, we describe a new class of protein disulfide isomerase (PDI) inhibitors that significantly and synergistically enhance the antitumor activity of HDACi in glioblastoma and pancreatic cancer preclinical models. RNA-sequencing screening coupled with gene silencing studies identified ATF3 as the driver of this antitumor synergy. ATF3 was highly induced by combined PDI and HDACi treatment as a result of increased acetylation of key histone lysine residues (acetylated histone 3 lysine 27 and histone 3 lysine 18) flanking the ATF3 promoter region. These chromatin marks were associated with increased RNA polymerase II recruitment to the ATF3 promoter, a synergistic upregulation of ATF3, and a subsequent apoptotic response in cancer cells. The HSP40/HSP70 family genes DNAJB1 and HSPA6 were found to be critical ATF3-dependent genes that elicited the antitumor response after PDI and HDAC inhibition. In summary, this study presents a synergistic antitumor combination of PDI and HDAC inhibitors and demonstrates a mechanistic and tumor suppressive role of ATF3. Combined treatment with PDI and HDACi offers a dual therapeutic strategy in solid tumors and the opportunity to achieve previously unrealized activity of HDACi in oncology. SIGNIFICANCE: This study uses a first-in-class PDI inhibitor entering clinical development to enhance the effects of epigenetic drugs in some of the deadliest forms of cancer.

Tuning isoform selectivity and bortezomib sensitivity with a new class of alkenyl indene PDI inhibitor.

Protein disulfide isomerase (PDI, PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies, although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4, CHOP, and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors.

Human bone marrow activates the Akt pathway in metastatic prostate cells through transactivation of the alpha-platelet-derived growth factor receptor.

The factors regulating the bone tropism of disseminated prostate cancer cells are still vaguely defined. We report that prostate cancer cells that metastasize to the skeleton respond to human bone marrow with a robust stimulation of the phosphatidylinositol 3-kinase/Akt pathway, whereas prostate cells that lack bone-metastatic potential respond negligibly. The majority of this Akt activation is dependent on alpha-platelet-derived growth factor receptor (alpha-PDGFR) signaling, which was shown using the small-molecule inhibitor of PDGFR signaling AG1296. Low concentrations of PDGF-AA and PDGF-BB found in bone marrow aspirates, which were detected by ELISA, do not account for the high levels of alpha-PDGFR signaling. Additionally, neutralizing PDGF binding using a alpha-PDGFR-specific antibody (IMC-3G3) failed to produce a significant inhibition of bone marrow-induced Akt activation. However, the inhibitory effect of IMC-3G3 rivaled that of AG1296 when incubation was done under conditions that stimulated alpha-PDGFR internalization. We conclude that alpha-PDGFR is activated by multiple soluble factors contained within human bone marrow, in addition to its natural ligands, and this transactivation is dependent on receptor localization to the plasma membrane. Therefore, alpha-PDGFR expression may provide select prostate phenotypes with a growth advantage within the bone microenvironment.

Discovery platform for inhibitors of IgH gene enhancer activity.

Immunoglobulin heavy chain (IgH) translocations are common and early oncogenic events in B cell and plasma cell malignancies including B cell non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). IgH translocations bring oncogenes into close proximity with potent enhancer elements within the IgH locus, leading to oncogene up-regulation. As IgH enhancer activity is tightly controlled by B cell lineage-specific signaling and transcriptional networks, we hypothesized that IgH enhancers are potentially druggable targets/elements. To test this, we developed a molecular imaging-based high-throughput screening platform for discovering inhibitors of IgH enhancer-driven transcriptional activity. As proof of concept, we identified a low micromolar potency molecule (compound 30666) that inhibited immunoglobulin production by MM cells and blocked expression of an array of IgH translocation-induced oncogenes (CCND1, FGFR3/MMSET, and MYC) in MM and NHL cell lines. Prolonged exposure to 30666 significantly reduced the viability of IgH translocation-positive NHL and MM cells, but was less effective against cells lacking IgH translocations. Compound 30666 exhibited suitable pharmacological properties, including metabolic stability in liver microsomes and oral bioavailability in mice, and demonstrated preclinical anti-MM activity in a plasmacytoma mouse model. Our work suggests that IgH enhancers are attractive and potentially druggable targets for IgH translocation driven malignancies.

Endoplasmic Reticulum Protein Disulfide Isomerase Shapes T Cell Efficacy for Adoptive Cellular Therapy of Tumors.

Effective cancer therapies simultaneously restrict tumor cell growth and improve anti-tumor immune responses. Targeting redox-dependent protein folding enzymes within the endoplasmic reticulum (ER) is an alternative approach to activation of the unfolded protein response (UPR) and a novel therapeutic platform to induce malignant cell death. E64FC26 is a recently identified protein disulfide isomerase (PDI) inhibitor that activates the UPR, oxidative stress, and apoptosis in tumor cells, but not normal cell types. Given that targeting cellular redox homeostasis is a strategy to augment T cell tumor control, we tested the effect of E64FC26 on healthy and oncogenic T cells. In stark contrast to the pro-UPR and pro-death effects we observed in malignant T cells, we found that E64FC26 improved viability and limited the UPR in healthy T cells. E64FC26 treatment also diminished oxidative stress and decreased global PDI expression in normal T cells. Oxidative stress and cell death are limited in memory T cells and we found that PDI inhibition promoted memory traits and reshaped T cell metabolism. Using adoptive transfer of tumor antigen-specific CD8 T cells, we demonstrate that T cells activated and expanded in the presence of E64FC26 control tumor growth better than vehicle-matched controls. Our data indicate that PDI inhibitors are a new class of drug that may dually inhibit tumor cell growth and improve T cell tumor control.

Inhibitors of the protein disulfide isomerase family for the treatment of multiple myeloma.

Multiple Myeloma (MM) is highly sensitive to disruptions in cellular protein homeostasis. Proteasome inhibitors (PIs) are initially effective in the treatment of MM, although cures are not achievable and the emergence of resistance limits the durability of responses. New therapies are needed for refractory patients, and those that combat resistance to standard of care agents would be particularly valuable. Screening of multiple chemical libraries for PI re-sensitizing compounds identified E61 as a potent enhancer of multiple PIs and MM specific activity. Using a tandem approach of click chemistry and peptide mass fingerprinting, we identified multiple protein disulfide isomerase (PDI) family members as the primary molecular targets of E61. PDIs mediate oxidative protein folding, and E61 treatment induced robust ER and oxidative stress responses as well as the accumulation of ubiquitinylated proteins. A chemical optimization program led to a new structural class of indene (exemplified by lead E64FC26), which are highly potent pan-style inhibitors of PDIs. In mice with MM, E64FC26 improved survival and enhanced the activity of bortezomib without any adverse effects. This work demonstrates the potential of E64FC26 as an early drug candidate and the strategy of targeting multiple PDI isoforms for the treatment of refractory MM and beyond.

Glutaminase inhibitor CB-839 synergizes with carfilzomib in resistant multiple myeloma cells.

Curative responses in the treatment of multiple myeloma (MM) are limited by the emergence of therapeutic resistance. To address this problem, we set out to identify druggable mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib), which are cornerstone agents in the treatment of MM. In isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. PI resistant cells exhibited increased mitochondrial respiration driven by glutamine as the principle fuel source. To target glutamine-induced respiration in PI resistant cells, we utilized the glutaminase-1 inhibitor, CB-839. CB-839 inhibited mitochondrial respiration and was more cytotoxic in PI resistant cells as a single agent. Furthermore, we found that CB-839 synergistically enhanced the activity of multiple PIs with the most dramatic synergy being observed with carfilzomib (Crflz), which was confirmed in a panel of genetically diverse PI sensitive and resistant MM cells. Mechanistically, CB-839 enhanced Crflz-induced ER stress and apoptosis, characterized by a robust induction of ATF4 and CHOP and the activation of caspases. Our findings suggest that the acquisition of PI resistance involves adaptations in cellular bioenergetics, supporting the combination of CB-839 with Crflz for the treatment of refractory MM.

Bone-metastatic potential of human prostate cancer cells correlates with Akt/PKB activation by alpha platelet-derived growth factor receptor.

Prostate adenocarcinoma metastasizes to the skeleton more frequently than any other organ. An underlying cause of this phenomenon may be the ability of bone-produced factors to specifically select disseminated prostate cancer cells that are susceptible to their trophic effects. Platelet-derived growth factor (PDGF), a potent mitogen for both normal and tumor cells, is produced in several tissues including bone, where it is synthesized by both osteoblasts and osteoclasts. Here, we show that PDGF causes a significantly stronger activation of the Akt/PKB survival pathway in bone-metastatic prostate cancer cells compared to nonmetastatic cells. Normal prostate epithelial cells and DU-145 prostate cells, originally derived from a brain metastasis, are not responsive to PDGF. In contrast, epidermal growth factor stimulates Akt to the same extent in all prostate cells tested. This difference in PDGF responsiveness depends on the higher expression of alpha-PDGFR in bone-metastatic compared to nonmetastatic prostate cells and the lack of alpha-PDGFR expression in normal and metastatic prostate cells derived from tissues other than bone. Thus, alpha-PDGFR expression might identify prostate cancer cells with the highest propensity to metastasize to the skeleton.

CX3CR1-fractalkine expression regulates cellular mechanisms involved in adhesion, migration, and survival of human prostate cancer cells.

Chemokines and their receptors might be involved in the selection of specific organs by metastatic cancer cells. For instance, the CXCR4-SDF-1alpha pair regulates adhesion and migration of breast as well as prostate cancer cells to metastatic sites. In this study, we present the first evidence for the expression of CX3CR1--the specific receptor for the chemokine fractalkine--by human prostate cancer cells, whereas human bone marrow endothelial cells and differentiated osteoblasts express fractalkine. The adhesion of prostate cancer cells to human bone marrow endothelial cells in flow conditions is significantly reduced by a neutralizing antibody against fractalkine, and they migrate toward a medium conditioned by osteoblasts, which secrete the soluble form of the chemokine. Finally, fractalkine activates the PI3K/Akt survival pathway in human prostate cancer cells.

Emerging Therapeutic Strategies for Overcoming Proteasome Inhibitor Resistance.

The debut of the proteasome inhibitor bortezomib (Btz; Velcade®) radically and immediately improved the treatment of multiple myeloma (MM), an incurable malignancy of the plasma cell. Therapeutic resistance is unavoidable, however, and represents a major obstacle to maximizing the clinical potential of the drug. To address this challenge, studies have been conducted to uncover the molecular mechanisms driving Btz resistance and to discover new targeted therapeutic strategies and combinations that restore Btz activity. This review discusses the literature describing molecular adaptations that confer Btz resistance with a primary disease focus on MM. Also discussed are the most recent advances in therapeutic strategies that overcome resistance, approaches that include redox-modulating agents, murine double minute 2 inhibitors, therapeutic monoclonal antibodies, and new epigenetic-targeted drugs like bromodomain and extra terminal domain inhibitors.

REDOX IMAGING OF THE p53-DEPENDENT MITOCHONDRIAL REDOX STATE IN COLON CANCER EX VIVO.

The mitochondrial redox state and its heterogeneity of colon cancer at tissue level have not been previously reported. Nor has how p53 regulates mitochondrial respiration been measured at (deep) tissue level, presumably due to the unavailability of the technology that has sufficient spatial resolution and tissue penetration depth. Our prior work demonstrated that the mitochondrial redox state and its intratumor heterogeneity is associated with cancer aggressiveness in human melanoma and breast cancer in mouse models, with the more metastatic tumors exhibiting localized regions of more oxidized redox state. Using the Chance redox scanner with an in-plane spatial resolution of 200 µm, we imaged the mitochondrial redox state of the wild-type p53 colon tumors (HCT116 p53 wt) and the p53-deleted colon tumors (HCT116 p53-/-) by collecting the fluorescence signals of nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins [Fp, including flavin adenine dinucleotide (FAD)] from the mouse xenografts snap-frozen at low temperature. Our results show that: (1) both tumor lines have significant degree of intratumor heterogeneity of the redox state, typically exhibiting a distinct bi-modal distribution that either correlates with the spatial core-rim pattern or the "hot/cold" oxidation-reduction patches; (2) the p53-/- group is significantly more heterogeneous in the mitochondrial redox state and has a more oxidized tumor core compared to the p53 wt group when the tumor sizes of the two groups are matched; (3) the tumor size dependence of the redox indices (such as Fp and Fp redox ratio) is significant in the p53-/- group with the larger ones being more oxidized and more heterogeneous in their redox state, particularly more oxidized in the tumor central regions; (4) the H&E staining images of tumor sections grossly correlate with the redox images. The present work is the first to reveal at the submillimeter scale the intratumor heterogeneity pattern of the mitochondrial redox state in colon cancer and the first to indicate that at tissue level the mitochondrial redox state is p53 dependent. The findings should assist in our understanding on colon cancer pathology and developing new imaging biomarkers for clinical applications.