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Material and Methods, Figure legends

Phenotypic, transcriptomic and genetic validation of PDX models

List of the SNVs identified through RNA-sequencing

Differentially expressed genes in the human B-ALL cohort

Characteristics of the B-ALL cohort

Reagents and Resources

Purpose:Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL.Experimental Design:To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents.Results:Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRASG12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine.Conclusions:Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia.

List of the SNVs identified through whole exome sequencing

List of fusion transcripts identified through RNA-sequencing

List of datasets enriched in murine B cell precursors (GSEA analyses)

The bradykinin receptor B1R is overexpressed in many human cancers where it might be used as a general target for cancer imaging. In this study, we evaluated the feasibility of using radiolabeled kallidin derivatives to visualize B1R expression in a preclinical model of B1R-positive tumors. Three synthetic derivatives were evaluated in vitro and in vivo for receptor binding and their ability to visualize tumors by PET. Enalaprilat and phosphoramidon were used to evaluate the impact of peptidases on tumor visualization. While we found that radiolabeled peptides based on the native kallidin sequence were ineffective at visualizing B1R-positive tumors, peptidase inhibition with phosphoramidon greatly enhanced B1R visualization in vivo. Two stabilized derivatives incorporating unnatural amino acids (68Ga-SH01078 and 68Ga-P03034) maintained receptor-binding affinities that were effective, allowing excellent tumor visualization, minimal accumulation in normal tissues, and rapid renal clearance. Tumor uptake was blocked in the presence of excess competitor, confirming that the specificity of tumor accumulation was receptor mediated. Our results offer a preclinical proof of concept for noninvasive B1R detection by PET imaging as a general tool to visualize many human cancers. Cancer Res; 75(2); 387–93. ©2014 AACR.

Contains fulltext : 241347.pdf (Publisher’s version ) (Closed access) Asparaginase (ASNase) therapy has been a mainstay of acute lymphoblastic leukemia (ALL) protocols for decades and shows promise in the treatment of a variety of other cancers. To improve the efficacy of ASNase treatment, we used a CRISPR/Cas9-based screen to identify actionable signaling intermediates that improve the response to ASNase. Both genetic inactivation of Bruton's tyrosine kinase (BTK) and pharmacological inhibition by the BTK inhibitor ibrutinib strongly synergize with ASNase by inhibiting the amino acid response pathway, a mechanism involving c-Myc-mediated suppression of GCN2 activity. This synthetic lethal interaction was observed in 90% of patient-derived xenografts, regardless of the genomic subtype. Moreover, ibrutinib substantially improved ASNase treatment response in a murine PDX model. Hence, ibrutinib may be used to enhance the clinical efficacy of ASNase in ALL. This trial was registered at www.clinicaltrials.gov as # NCT02884453.

DYRK1A is a serine/threonine kinase encoded on human chromosome 21 (HSA21) that has been implicated in several pathologies of Down syndrome (DS), including cognitive deficits and Alzheimer's disease. Although children with DS are predisposed to developing leukemia, especially B cell acute lymphoblastic leukemia (B-ALL), the HSA21 genes that contribute to malignancies remain largely undefined. Here, we report that DYRK1A is overexpressed and required for B-ALL. Genetic and pharmacologic inhibition of DYRK1A decreased leukemic cell expansion and suppressed B-ALL development in vitro and in vivo. Furthermore, we found that FOXO1 and STAT3, transcription factors that are indispensable for B cell development, are critical substrates of DYRK1A. Loss of DYRK1A-mediated FOXO1 and STAT3 signaling disrupted DNA damage and ROS regulation, respectively, leading to preferential cell death in leukemic B cells. Thus, we reveal a DYRK1A/FOXO1/STAT3 axis that facilitates the development and maintenance of B-ALL.

Despite rapid progress in genomic profiling in acute lymphoblastic leukemia (ALL), identification of actionable targets and prediction of response to drugs remains challenging. To identify specific vulnerabilities in ALL, we performed a drug screen using primary human ALL samples cultured in a model of the bone marrow microenvironment combined with high content image analysis. Among the 2487 FDA-approved compounds tested, anthelmintic agents of the class of macrocyclic lactones exhibited potent anti-leukemia activity, similar to the already known anti-leukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and T-ALL including refractory relapse cases confirmed strong anti-leukemia activity with IC50 values in the low micromolar range. Anthelmintic agents increased intracellular chloride levels in primary leukemia cells, inducing mitochondrial outer membrane depolarization and cell death. Supporting the notion that simultaneously targeting cell death machineries at different angles may enhance the cell death response, combination of anthelmintic agents with the BCL-2 antagonist navitoclax or with the chemotherapeutic agent dexamethasone showed synergistic activity in primary ALL. These data reveal anti-leukemia activity of anthelmintic agents and support exploiting drug repurposing strategies to identify so far unrecognized anti-cancer agents with potential to eradicate even refractory leukemia.

Despite major advances in the treatment of patients with acute lymphoblastic leukemia in the last decades, refractory and/or relapsed disease remains a clinical challenge, and relapsed leukemia patients have an exceedingly dismal prognosis. Dysregulation of apoptotic cell death pathways is a leading cause of drug resistance; thus, alternative cell death mechanisms, such as necroptosis, represent an appealing target for the treatment of high-risk malignancies. We and other investigators have shown that activation of receptor interacting protein kinase 1 (RIP1)–dependent apoptosis and necroptosis by second mitochondria derived activator of caspase mimetics (SMs) is an attractive antileukemic strategy not currently exploited by standard chemotherapy. However, the underlying molecular mechanisms that determine sensitivity to SMs have remained elusive. We show that tumor necrosis factor receptor 2 (TNFR2) messenger RNA expression correlates with sensitivity to SMs in primary human leukemia. Functional genetic experiments using clustered regularly interspaced short palindromic repeats/Cas9 demonstrate that TNFR2 and TNFR1, but not the ligand TNF-α, are essential for the response to SMs, revealing a ligand-independent interplay between TNFR1 and TNFR2 in the induction of RIP1-dependent cell death. Further potential TNFR ligands, such as lymphotoxins, were not required for SM sensitivity. Instead, TNFR2 promotes the formation of a RIP1/TNFR1-containing death signaling complex that induces RIP1 phosphorylation and RIP1-dependent apoptosis and necroptosis. Our data reveal an alternative paradigm for TNFR2 function in cell death signaling and provide a rationale to develop strategies for the identification of leukemias with vulnerability to RIP1-dependent cell death for tailored therapeutic interventions.

Despite progress with immunotherapy and targeted agents, treatment of refractory disease remains challenging, in particular for patients with T-ALL. Identification of genomic lesions defining actionable targets had limited impact on patient care so far. The complexity of biological systems highlights the need to develop complementary functional approaches. We and others have established platforms with a library of 120 drugs based on current treatment and (pre-)clinical development, to detect ex-vivo drug response phenotypes on leukemia samples at single cell resolution by high content image analysis. We demonstrated that drug response profiling (DRP) identifies dependencies not predicted by genetic alterations adding a functional information layer for clinicians. Here we report first correlations with clinical outcome using DRP in a non-interventional setting. From 2016 to 2019 we performed DRP in the framework of European ALL first- and second- line protocols upon request by treating centers. Here we analyze retrospectively treatment decisions and outcome for 23 T- and 50 BCP-ALL patients. To evaluate drug responses, we compared dose response curves of individual patients to data recorded for all patients. Sensitivity and resistance were defined based on the IC50 outlier analysis using cut-offs depending on distribution (normal gaussian vs. skewed). From 73 patients tested, clinical outcome data has been available for 36 BCP- and 15 T-ALL patients. NGS data provided by the INFORM registry has been available in 8 BCP- and 2 T-ALL patients. In first line BCP-ALL patients, ex-vivo Dexamethasone response predicted clinical response to prephase prednisone (d8) and minimal residual disease (MRD) reduction measured by flow cytometry at d15 of first line AIEOP BFM 2009 induction (Fig. 1). For refractory and relapsed ALL we observed an association of DRP and response to targeted agents in 14 out of 16 patients (87.5 %; Table 1). Data for the r/r BCP-ALL cohort is limited because most patients underwent CD19 and / or CD22-directed immunotherapy. Sensitivity and resistance to Calicheamicin correlated with clinical response to Inotuzumab, suggesting functional testing to be evaluated in future studies. In contrast, lack of correlation of ex-vivo sensitivity to MEK-inhibitors with presence of RAS-pathway alterations caution the exclusive use of molecular information to predict response to these agents. Most therapeutic decisions based on DRP information were made for patients with r/r T-ALL. Bortezomib ex-vivo sensitivity correlated with clinical responses in 5 T-ALL patients (Fig. 2). Both patients predicted to respond to Bortezomib and treated on Bortezomib + Venetoclax experienced good MRD response providing a bridge to stem cell transplantation (SCT). However, these patients relapsed after SCT emphasizing the need for additional consolidative therapeutic elements for heavily pretreated patients. In line with previous reports, we confirmed a T-ALL with high sensitivity to Dasatinib (IC50 1.9 nM; Fig. 3). Dasatinib monotherapy induced a molecular remission. A 2nd T-ALL showing an ex- vivo Dasatinib IC50 at 80 nM was refractory to treatment with Dasatinib + Daunoxome-FLAG. A 3rd ABL1-fusion positive T-ALL, ex-vivo resistant to Imatinib and Dasatinib, had only short- term response to Imatinib + chemotherapy. Finally, treating a T-ALL patient based on high sensitivity to the XPO1 inhibitor Selinexor as 4th line monotherapy led to significant decrease of PB blasts from d1 25 G/L to 0.7 G/L at d13 of treatment (Fig. 4). The patient experienced improved quality of life, minimizing need of hospitalization with stable disease for 3 months on maintenance with Selinexor. Given the promising preclinical data with this class of agents and current lack of established biomarkers, we propose that DRP should be evaluated for this class of agents. In conclusion, we established first associations between DRP and clinical response for various agents providing a rationale for the evaluation of DRP in prospective clinical trials. Integration of molecular and functional information may improve the selection of more specific treatment options for patients with resistant disease. The international BFM Study Group and ITCC Consortium are planning an international multiarm early clinical trial for treatment of r/r ALL patients that will include DRP for evaluation in order to improve selection of targeted therapy. Disclosures Cario: Jazz Pharmaceuticals: Consultancy, Other: travel support; Novartis: Consultancy, Other: travel support. Hrusak:Amgen: Other: MRD investigations funded by Amgen, Research Funding. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. von Stackelberg:Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau. Jacoby:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lonza: Membership on an entity's Board of Directors or advisory committees. Bourquin:Servier: Other: Travel Support.

Deregulated cell death pathways contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Intrinsic apoptosis signaling is regulated by different proapoptotic and antiapoptotic molecules: proapoptotic BCL-2 homology domain 3 (BH3) proteins activate prodeath molecules leading to cellular death, while antiapoptotic molecules including B-cell lymphoma 2 (BCL-2) prevent activation of prodeath proteins and counter-regulate apoptosis induction. Inhibition of these antiapoptotic regulators has become a promising strategy for anticancer treatment, but variable anticancer activities in different malignancies indicate the need for upfront identification of responsive patients. Here, we investigated the activity of the BCL-2 inhibitor venetoclax (VEN, ABT-199) in B-cell precursor acute lymphoblastic leukemia and found heterogeneous sensitivities in BCP-ALL cell lines and in a series of patient-derived primografts. To identify parameters of sensitivity and resistance, we evaluated genetic aberrations, gene-expression profiles, expression levels of apoptosis regulators, and functional apoptosis parameters analyzed by mitochondrial profiling using recombinant BH3-like peptides. Importantly, ex vivo VEN sensitivity was most accurately associated with functional BCL-2 dependence detected by BH3 profiling. Modeling clinical application of VEN in a preclinical trial in a set of individual ALL primografts, we identified that leukemia-free survival of VEN treated mice was precisely determined by functional BCL-2 dependence. Moreover, the predictive value of ex vivo measured functional BCL-2 dependence for preclinical in vivo VEN response was confirmed in an independent set of primograft ALL including T- and high risk-ALL. Thus, integrative analysis of the apoptosis signaling indicating mitochondrial addiction to BCL-2 accurately predicts antileukemia activity of VEN, robustly identifies VEN-responsive patients, and provides information for stratification and clinical guidance in future clinical applications of VEN in patients with ALL.

Caspase-3 and -7 are redundantly required to amplify the extrinsic and intrinsic apoptotic cascade in human leukemia., Apoptosis is a complex multi-step process driven by caspase-dependent proteolytic cleavage cascades. Dysregulation of apoptosis promotes tumorigenesis and limits the efficacy of chemotherapy. To assess the complex interactions among caspases during apoptosis, we disrupted caspase-8, -9, -3, -7, or -6 and combinations thereof, using CRISPR-based genome editing in living human leukemia cells. While loss of apical initiator caspase-8 or -9 partially blocked extrinsic or intrinsic apoptosis, respectively, only combined loss of caspase-3 and -7 fully inhibited both apoptotic pathways, with no discernible effect of caspase-6 deficiency alone or in combination. Caspase-3/7 double knockout cells exhibited almost complete inhibition of caspase-8 or -9 activation. Furthermore, deletion of caspase-3 and -7 decreased mitochondrial depolarization and cytochrome c release upon apoptosis activation. Thus, activation of effector caspase-3 or -7 sets off explosive feedback amplification of upstream apoptotic events, which is a key feature of apoptotic signaling essential for efficient apoptotic cell death.

ispartof: HAEMATOLOGICA vol:104 issue:1 pages:E17-E20 ispartof: location:Italy status: published

Two fluorine-18 ((18)F) labeled bradykinin B1 receptor (B1R)-targeting small molecules, (18)F-Z02035 and (18)F-Z02165, were synthesized and evaluated for imaging with positron emission tomography (PET). Z02035 and Z02165 were derived from potent antagonists, and showed high binding affinity (0.93±0.44 and 2.80±0.50nM, respectively) to B1R. (18)F-Z02035 and (18)F-Z02165 were prepared by coupling 2-[(18)F]fluoroethyl tosylate with their respective precursors, and were obtained in 10±5 (n=4) and 22±14% (n=3), respectively, decay-corrected radiochemical yield with >99% radiochemical purity. (18)F-Z02035 and (18)F-Z02165 exhibited moderate lipophilicity (LogD7.4=1.10 and 0.59, respectively), and were stable in mouse plasma. PET imaging and biodistribution studies in mice showed that both tracers enabled visualization of the B1R-positive HEK293T::hB1R tumor xenografts with better contrast than control B1R-negative HEK293T tumors. Our data indicate that small molecule antagonists can be used as pharmacophores for the design of B1R-targeting PET tracers.

Bradykinin B1 receptor (B1R), which is upregulated in a variety of malignancies, is an attractive cancer imaging biomarker. In this study we optimized the selection of radiolabel-chelator complex to improve tumor uptake and tumor-to-background contrast of radiolabeled analogues of B9958 (Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-d-Tic-Cpg), a potent B1R antagonist. Peptide sequences were assembled on solid phase. Cold standards were prepared by incubating DOTA-/NODA-conjugated peptides with GaCl3, and by incubating AlOH-NODA-conjugated peptide with NaF. Binding affinities were measured via in vitro competition binding assays. (68)Ga and (18)F labeling experiments were performed in acidic buffer and purified by HPLC. Imaging/biodistribution studies were performed in mice bearing both B1R-positive (B1R+) HEK293T::hB1R and B1R-negative (B1R-) HEK293T tumors. Z02176 (Ga-DOTA-Pip-B9958; Pip: 4-amino-(1-carboxymethyl)piperidine), Z02137 (Ga-NODA-Mpaa-Pip-B9958; Mpaa: 4-methylphenylacetic acid), and Z04139 (AlF-NODA-Mpaa-Pip-B9958) bound hB1R with high affinity (Ki = 1.4-2.5 nM). (68)Ga-/(18)F-labeled peptides were obtained on average in ≥32% decay-corrected radiochemical yield with99% radiochemical purity and 100-261 GBq/μmol specific activity. Biodistribution/imaging studies at 1 h postinjection showed that all tracers cleared rapidly from background tissues (except kidneys) and were excreted predominantly via the renal pathway. Only kidneys, bladders, and B1R+ tumors were clearly visualized in PET images. Uptake in B1R+ tumor was higher by using (68)Ga-Z02176 (28.9 ± 6.21 %ID/g) and (18)F-Z04139 (22.6 ± 3.41 %ID/g) than (68)Ga-Z02137 (14.0 ± 4.86 %ID/g). The B1R+ tumor-to-blood and B1R+ tumor-to-muscle contrast ratios were also higher for (68)Ga-Z02176 (56.1 ± 17.3 and 167 ± 57.6) and (18)F-Z04139 (58.0 ± 20.9 and 173 ± 42.9) than (68)Ga-Z02137 (34.3 ± 15.2 and 103 ± 30.2). With improved target-to-background contrast (68)Ga-Z02176 and (18)F-Z04139 are promising for imaging B1R expression in cancers with PET.

Bradykinin B1 receptor (B1R) that is overexpressed in cancers but minimally expressed in normal healthy tissues represents an attractive biomarker for the development of cancer imaging agents. The goal of this study was to evaluate the effect of different linkers on the pharmacokinetics and tumor uptake of a B1R-targeting radio-peptide sequence, 68Ga-DOTA-linker-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu. Four peptides, SH01078, P03034, P04115, and P04168, with 6-aminohexanoic acid, 9-amino-4,7-dioxanonanoic acid, Gly-Gly, and 4-amino-(1-carboxymethyl)piperidine, respectively, as the linker were synthesized and evaluated. In vitro competition binding assays showed that the Ki values of SH01078, P03034, P04115, and P04168 were 27.8±4.9, 16.0±1.9, 11.4±2.5, and 3.6±0.2 nM, respectively. Imaging and biodistribution studies were performed in mice bearing both B1R-positive HEK293T::hB1R and B1R-negative HEK293T tumors. All tracers showed mainly renal excretion with excellent tumor visualization and minimal background activity except for kidneys and bladder. The average uptake of 68Ga-labeled SH01078, P03034, and P04115 in HEK293T::hB1R tumor was similar (1.96-2.17%ID/g) at 1 h postinjection. 68Ga-P04168 generated higher HEK293T::hB1R tumor uptake (4.15±1.13%ID/g) and lower background activity, leading to a2-fold improvement in HEK293T::hB1R tumor-to-background (HEK293T tumor, blood, muscle, and liver) contrasts over those of 68Ga-labeled SH01078, P03034, and P04115. Our results indicate that the choice of linker affects binding affinity, pharmacokinetics, and tumor targeting. The use of the cationic 4-amino-(1-carboxymethyl)piperidine linker improved tumor visualization, and the resulting 68Ga-P04168 might be promising for clinical application for imaging B1R-expressing tumors with positron emission tomography.

Bradykinin B1 receptor (B1R) is a G-protein–coupled receptor that is overexpressed in a variety of cancers. B1R is not expressed in healthy tissues, making it an attractive cancer imaging marker. Previously, we reported selective uptake of 68Ga-P03034 (68Ga-DOTA-dPEG2-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu) in B1R-positive (B1R+) HEK293T::hB1R tumor xenografts in mice. In this study, we compare 68Ga-P03034 with 68Ga-labeled P04158 (68Ga-DOTA-dPEG2-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic) and Z02090 (68Ga-DOTA-dPEG2-Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-D-Tic-Cpg) derived from 2 potent B1R antagonists, B9858 and B9958, respectively, for imaging B1R expression with PET. Methods: Peptide sequences were assembled on solid-phase. Cold standards were prepared by incubating DOTA-conjugated peptides with GaCl3. Binding affinity was measured via competition binding assays using hB1R-expressing Chinese hamster ovary-K1 cell membranes. 68Ga labeling was performed in N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) buffer with microwave heating and purified by high-performance liquid chromatography. Imaging/biodistribution studies were performed in mice bearing wild-type HEK293T (B1R−) and B1R+ HEK293T::hB1R tumors. Results: P03034, P04158, and Z02090 bound B1R with high affinity, with Ki values at 16.0 ± 2.9, 1.5 ± 1.9, and 1.1 ± 0.8 nM, respectively. 68Ga-labeled P03034, P04159, and Z02090 were obtained in greater than 50% decay-corrected radiochemical yields with more than 99% radiochemical purity. Biodistribution studies showed that all three 68Ga-labeled tracers cleared rapidly from the blood and normal tissues, with excretion mainly via the renal pathway. At 1 h after injection, only the kidneys, bladders, and B1R+ HEK293T::hB1R tumors were clearly visualized in PET images. Uptake values of 68Ga-labeled P03034, P04158, and Z02090 in B1R+ tumors were 2.17 ± 0.49, 19.6 ± 4.50, and 14.4 ± 1.63 percentage injected dose per gram, respectively. Uptake ratios of B1R+ to B1R− tumor, blood, and muscle were 6.23 ± 1.69, 5.72 ± 2.20, and 25.5 ± 13.1 for 68Ga-P03034; 34.5 ± 10.5, 19.2 ± 8.21, and 66.1 ± 17.0 for 68Ga-P04158; and 29.3 ± 9.68, 29.9 ± 5.58, and 124 ± 28.1 for 68Ga-Z02090, respectively. Conclusion: All three 68Ga-labeled B1R-targeting peptides generated specific and high-contrasted images of B1R+ tumors xenografted in mice. With significantly higher tumor uptake and target-to-nontarget ratios, 68Ga-labeled P04158 and Z02090 are superior to P03034 for imaging B1R expression with PET.

The bradykinin receptor B1R is overexpressed in many human cancers where it might be used as a general target for cancer imaging. In this study, we evaluated the feasibility of using radiolabeled kallidin derivatives to visualize B1R expression in a preclinical model of B1R-positive tumors. Three synthetic derivatives were evaluated in vitro and in vivo for receptor binding and their ability to visualize tumors by PET. Enalaprilat and phosphoramidon were used to evaluate the impact of peptidases on tumor visualization. While we found that radiolabeled peptides based on the native kallidin sequence were ineffective at visualizing B1R-positive tumors, peptidase inhibition with phosphoramidon greatly enhanced B1R visualization in vivo. Two stabilized derivatives incorporating unnatural amino acids (68Ga-SH01078 and 68Ga-P03034) maintained receptor-binding affinities that were effective, allowing excellent tumor visualization, minimal accumulation in normal tissues, and rapid renal clearance. Tumor uptake was blocked in the presence of excess competitor, confirming that the specificity of tumor accumulation was receptor mediated. Our results offer a preclinical proof of concept for noninvasive B1R detection by PET imaging as a general tool to visualize many human cancers. Cancer Res; 75(2); 387–93. ©2014 AACR.

Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.

The cystine transporter (system xC−) is an antiporter of cystine and glutamate. It has relatively low basal expression in most tissues and becomes upregulated in cells under oxidative stress (OS) as one of the genes expressed in response to the antioxidant response element promoter. We have developed 18F-5-fluoroaminosuberic acid (FASu), a PET tracer that targets system xC−. The goal of this study was to evaluate 18F-FASu as a specific gauge for system xC− activity in vivo and its potential for breast cancer imaging. Methods: 18F-FASu specificity toward system xC− was studied by cell inhibition assay, cellular uptake after OS induction with diethyl maleate, with and without anti-xCT small interfering RNA knockdown, in vitro uptake studies, and in vivo uptake in a system xC−–transduced xenograft model. In addition, radiotracer uptake was evaluated in 3 breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1. Results: Reactive oxygen species–inducing diethyl maleate increased glutathione levels and 18F-FASu uptake, whereas gene knockdown with anti-xCT small interfering RNA led to decreased tracer uptake. 18F-FASu uptake was robustly inhibited by system xC− inhibitors or substrates, whereas uptake was significantly higher in transduced cells and tumors expressing xCT than in wild-type HEK293T cells and tumors (P < 0.0001 for cells, P = 0.0086 for tumors). 18F-FASu demonstrated tumor uptake in all 3 breast cancer cell lines studied. Among them, triple-negative breast cancer MDA-MB-231, which has the highest xCT messenger RNA level, had the highest tracer uptake (P = 0.0058 when compared with MCF-7; P < 0.0001 when compared with ZR-75-1). Conclusion: 18F-FASu as a system xC− substrate is a specific PET tracer for functional monitoring of system xC− and OS imaging. By enabling noninvasive analysis of xC− responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.

Introduction: Despite the improvements, still too many patients die for their lymphomas and novel compounds are needed. We present a new small molecule, EG-011 (PCT/EP2018/057678), with in vitro and in vivo anti-cancer activity in lymphoma models. Methods: Lymphoma and solid tumor cell lines were exposed to a large range of concentrations of EG-011 as single agent for 72h, followed by MTT proliferation assay and IC50 calculation. Cell viability of twelve acute lymphoblastic leukemia (ALL) primary patient cells from different high-risk subgroups (VNN2+, E2A-HLF, refractory T and IKZF plus) co-culture with marrow-derived MSCs were assayed after 72h of incubation with EG-011 and controls. Apoptosis assay was measured with annexin V by FACS. Xenografts were established s.c. into the left flanks of female NOD-SCID mice; treatment (200 mg/kg, i.p. 5 days per week) started with already established tumors. Combinations were evaluated with Chou-Talalay combination index (CI): synergism ( 1.1) after 72 hr treatments. Results: EG-011 presented a median IC50 of 2.25 μM in 62 lymphoma cell lines (95% C.I. 1-5μM). A higher activity was observed in a group of 21 cell lines that had a median IC50 of 250 nM (95% C.I. 40-600 nM). Among these there were 11 germinal center B cell (GCB) diffuse large B cell lymphomas (DLBCL) (sensitive n=11/21, resistant n=9/41, P < 0.05), 4 mantle cell lymphoma (MCL) (sensitive n=4/21, resistant n=6/41, P n.s.), 3 marginal zone lymphoma (sensitive n=3/21, resistant n=2/41, P n.s.). EG-011 did not show any anti-proliferative activity in a panel of 25 solid tumor cell lines (IC50s > 10 μM), Among 12 primary ALL samples, 7 were sensitive to EG-011 with IC50 values between 0.3-4.6 µM after 72h, 5 displayed IC50 higher than 20 µM. A dose-dependent increase in cell death (20-55%) was observed in lymphoma cell lines (OCI-LY-19 and REC1) (500 nM and 2 μM; 72h). No cytotoxicity was seen in PBMCs from two healthy donors after treatment at 1 and 10 μM for 24h and 48h. In an in vivo xenograft experiment with the MCL REC-1 cell line, EG-011 delayed tumor growth (Day 6, Day 7, Day 9, P < 0.05) and tumor weight. EG-011-treated tumors were 2.2-fold smaller than controls (P < 0.001). Combinations were tested in DLBCL (OCI-LY-1, OCI-LY-8, TMD8) and MCL (REC1, MINO). EG-011 was synergistic with rituximab, bendamustine, venetoclax, ibrutinib and lenalidomide in all tested cell lines. Conclusion: The selective anti-lymphoma activity, in both in vitro and in vivo models, and the observed in vitro synergisms with FDA approved targeted agents make EG-011 a novel intriguing new drug candidate deserving further preclinical studies. Citation Format: Eugenio Gaudio, Filippo Spriano, Chiara Tarantelli, Matilde Guala, Eugenia Riveiro, Gaetanina Golino, Antonio Lupia, Giosuè Costa, Roberta Rocca, Luciano Cascione, Silvia Jenni, Yi-Chien Tsai, Beat Bornhauser, Stefano Alcaro, Francesco Paduano, Francesco Trapasso, Emanuele Zucca, Anastasios Stathis, Natalina Pazzi, Franco Cavalli, Francesco Bertoni. EG-011 is a novel small molecule with in vitro and in vivo anti-tumor activity against lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4796.

A novel

The neuropeptide Y1 receptor (Y1R) is overexpressed in many human cancers, particularly breast cancer. Due to stability issues, limited success has been achieved for Y1R imaging agents, including full length and truncated neuropeptide Y (NPY) analogues. The goal of this study was to evaluate the possibility of using radiolabeled truncated NPY analogues to visualize Y1R expression in a preclinical model of Y1R-positive tumor. Four truncated NPY analogues were synthesized based on the sequence of [Pro30, Tyr32, Leu34]NPY(28–36), also known as BVD15. We substituted Tyr5 and Arg6 with unnatural amino acids aiming to enhance plasma stability while maintaining good receptor binding affinity to Y1R. In addition, we substituted Leu4 to Lys4 in order to conjugate via an optional linker the DOTA chelator for 68Ga labeling. Receptor binding affinity and plasma stability of these compounds were evaluated. Positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies were performed usin...

Carbonic anhydrase IX (CA-IX) is a HIF-1-inducible enzyme that is overexpressed in many cancer subtypes to promote survival and invasion in hypoxic niches. Pharmacologic inhibition of CA-IX is achievable through sulfonamide-based inhibitors and has been shown to reduce primary growth of cancers and distant metastasis in preclinical models. We explored a multivalent approach for targeting CA-IX in vivo, noninvasively, with positron emission tomography. Three (68)Ga-polyaminocarboxylate chelator complex-conjugated tracers containing one, two, or three 4-(2-aminoethyl)benzenesulfonamide moieties were synthesized and evaluated for protein binding and imaging properties. Binding affinity to CA-I, -II, -IX, and -XII were determined using a stopped-flow CA catalyzed CO2 hydration assay. Biodistribution and PET/CT imaging were performed using immunocompromised mice bearing CA-IX expressing HT-29 colorectal tumors. Compounds demonstrated good binding affinity to CA-IX (Ki: 7.7-25.4 nM). (68)Ga-labeled sulfonamides were obtained in 64-91% decay-corrected average radiochemical yields with 50-536 GBq/μmol specific activity and97% average radiochemical purity. All three tracers allowed for the visualization of tumor xenografts at 1 h postinjection, with the monomer displaying the highest contrast. Tumor uptake of the monomer was blockable in the presence of acetazolamide, confirming target specificity. The monomer was excreted predominantly through the kidneys, while the dimer and trimer were cleared by both renal and hepatobiliary pathways. According to biodistribution analysis, tumor uptake (%ID/g) of the monomeric, dimeric, and trimeric tracers were 0.81 ± 0.15, 1.93 ± 0.26, and 2.30 ± 0.53 at 1 h postinjection. This corresponded to tumor-to-muscle ratios of 5.02 ± 0.22, 4.07 ± 0.87, and 4.18 ± 0.84, respectively. Our data suggest that (68)Ga-polyaminocarboxylate chelator-conjugated sulfonamides can be used to noninvasively image CA-IX. These CA-IX targeting PET tracers may be used to identify patients who can benefit from treatments targeting this protein or serve as surrogate imaging agents for tumor hypoxia.

Four trifluoroborate derivatives of phosphonium cations 2a-d were radiolabeled with fluorine-18 ((18)F) and evaluated for imaging myocardial perfusion with positron emission tomography (PET). Tracers were radiolabeled simply via (18)F-(19)F isotope exchange reaction in acidic (pH 2) aqueous solution. On average, [(18)F]2a-d were obtained in 10-17% non-decay-corrected radiochemical yield with 25.9-48.1GBq/μmol specific activity, and96% radiochemical purity. In vitro stability study showed no decomposition of [(18)F]2a-d after being incubated in mouse plasma for up to 2h. Myocardial uptake in mice was visualized in PET images by using [(18)F]2b-d but not [(18)F]2a. [(18)F]2a-d were stable against in vivo defluorination as no significant bone uptake was observed. Despite sub-optimal heart uptake of [(18)F]2b-d, we successfully demonstrated that (18)F-(19)F isotope exchange reaction on trifluoroborates could be a promising strategy for the design of potential (18)F-labeled tracers even for intracellular targets.

Structural variation (SV), involving deletions, duplications, inversions and translocations of DNA segments, is a major source of genetic variability in somatic cells and can dysregulate cancer-related pathways. However, discovering somatic SVs in single cells has been challenging, with copy-number-neutral and complex variants typically escaping detection. Here we describe single-cell tri-channel processing (scTRIP), a computational framework that integrates read depth, template strand and haplotype phase to comprehensively discover SVs in individual cells. We surveyed SV landscapes of 565 single cells, including transformed epithelial cells and patient-derived leukemic samples, to discover abundant SV classes, including inversions, translocations and complex DNA rearrangements. Analysis of the leukemic samples revealed four times more somatic SVs than cytogenetic karyotyping, submicroscopic copy-number alterations, oncogenic copy-neutral rearrangements and a subclonal chromothripsis event. Advancing current methods, single-cell tri-channel processing can directly measure SV mutational processes in individual cells, such as breakage-fusion-bridge cycles, facilitating studies of clonal evolution, genetic mosaicism and SV formation mechanisms, which could improve disease classification for precision medicine.