CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

ABSTRACT: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

BTK inhibition sensitizes acute lymphoblastic leukemia to asparaginase by suppressing the amino acid response pathway.

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.

Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia.

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 BCL2-inhibitor 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.

Synthesis and evaluation of a 68Ga-labeled bradykinin B1 receptor agonist for imaging with positron emission tomography.

A novel 68Ga-labeled bradykinin B1 receptor (B1R) agonist, 68Ga-Z01115, was synthesized and evaluated for imaging with positron emission tomography (PET). Z01115 exhibited good binding affinity (Ki=25.4±5.1nM) to hB1R. 68Ga-Z01115 was prepared in 74±5 decay-corrected radiochemical yield with >99% radiochemical purity and 155±89GBq/µmol (4.2±2.4Ci/µmol) specific activity. 68Ga-Z01115 was stable in vitro in mouse plasma (93% remaining intact after 60min incubation), and relatively stable in vivo (51±5% remaining intact at 5min post-injection). PET imaging and biodistribution studies in mice showed that 68Ga-Z01115 cleared rapidly from nontarget tissues/organs, and generated high target-to-nontarget contrast images. The uptake of 68Ga-Z01115 in B1R-positive (B1R+) tumor was 5.65±0.59%ID/g at 1h post-injection. Average contrast ratios of B1R+ tumor-to-B1R- tumor, -to-blood and -to-muscle were 24.3, 24.4 and 82.9, respectively. Uptake of 68Ga-Z01115 in B1R+ tumors was reduced by ∼90% with co-injection of cold standard, confirming it was mediated by B1R. Our data suggest that 68Ga-Z01115 is a promising tracer for imaging the expression of B1R that is overexpressed in a variety of cancers.

Targeting the Neuropeptide Y1 Receptor for Cancer Imaging by Positron Emission Tomography Using Novel Truncated Peptides.

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 using immune-compromised mice bearing HEK293T::WT and HEK293T::hY1R tumors. [Lys(Ga-DOTA)4, Bip5]BVD15 (CCZ01035), [Lys(Ahx-Ga-DOTA)4, Bip5]BVD15 (CCZ01053), and [Lys(Pip-Ga-DOTA)4, Bip5]BVD15 (CCZ01055) demonstrated good binding affinity to Y1R (Ki = 23.4-32.3 nM), while [Lys(Ga-DOTA)4, Har6]BVD15 (P05067) showed poor binding affinity (Ki > 1000 nM). In addition, CCZ01055 exhibited low binding affinity (Ki > 1000 nM) to Y2R and Y4R, demonstrating its selectivity to Y1R. The former three peptides showed improved in vitro plasma stability of 7-16% remaining intact after 1 h incubation. PET/CT imaging and biodistribution studies for 68Ga-labeled CCZ01053, CCZ01035, and CCZ01055 showed that radioactivity was mainly cleared by the renal pathway, and HEK293T::hY1R tumors were clearly visualized with minimal background activity with the latter two. Of these two tracers, [68Ga]CCZ01055 provided lower kidney accumulation and higher contrast, i.e., average uptake ratios of Y1R tumor to wild type tumor, blood, and muscle are 3.87 ± 0.83, 4.12 ± 1.14, and 17.6 ± 4.64, respectively. Furthermore, Y1R tumor uptake with [68Ga]CCZ01055 was significantly reduced with coinjection of 100 µg of peptide YY, confirming the specificity of tumor accumulation was receptor mediated. We successfully developed the first Y1R-targeting truncated NPY analogues for PET imaging in a preclinical model, and [68Ga]CCZ01055 is a critical template for designing improved imaging agents to detect Y1R expressing cancers.

PET Imaging of Carbonic Anhydrase IX Expression of HT-29 Tumor Xenograft Mice with (68)Ga-Labeled Benzenesulfonamides.

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 and >97% 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.

Radiolabeled B9958 Derivatives for Imaging Bradykinin B1 Receptor Expression with Positron Emission Tomography: Effect of the Radiolabel-Chelator Complex on Biodistribution and Tumor Uptake.

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 with >99% 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.

Design, synthesis and evaluation of (18)F-labeled bradykinin B1 receptor-targeting small molecules for PET imaging.

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.

Synthesis and evaluation of (18)F-trifluoroborate derivatives of triphenylphosphonium for myocardial perfusion imaging.

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, and >96% 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.

Imaging Bradykinin B1 Receptor with 68Ga-Labeled [des-Arg10]Kallidin Derivatives: Effect of the Linker on Biodistribution and Tumor Uptake.

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 a >2-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.

DYRK1A regulates B cell acute lymphoblastic leukemia through phosphorylation of FOXO1 and STAT3.

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.

TNFR2 is required for RIP1-dependent cell death in human 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.

Repurposing anthelmintic agents to eradicate resistant leukemia.

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.

Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia.

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.

Single-cell analysis of structural variations and complex rearrangements with tri-channel processing.

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.

Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7.

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.

Prediction of venetoclax activity in precursor B-ALL by functional assessment of apoptosis signaling.

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.

The Leukemogenic TCF3-HLF Complex Rewires Enhancers Driving Cellular Identity and Self-Renewal Conferring EP300 Vulnerability.

The chimeric transcription factor TCF3-HLF defines an incurable acute lymphoblastic leukemia subtype. Here we decipher the regulome of endogenous TCF3-HLF and dissect its essential transcriptional components and targets by functional genomics. We demonstrate that TCF3-HLF recruits HLF binding sites at hematopoietic stem cell/myeloid lineage associated (super-) enhancers to drive lineage identity and self-renewal. Among direct targets, hijacking an HLF binding site in a MYC enhancer cluster by TCF3-HLF activates a conserved MYC-driven transformation program crucial for leukemia propagation in vivo. TCF3-HLF pioneers the cooperation with ERG and recruits histone acetyltransferase p300 (EP300), conferring susceptibility to EP300 inhibition. Our study provides a framework for targeting driving transcriptional dependencies in this fatal leukemia.

18F-5-Fluoroaminosuberic Acid as a Potential Tracer to Gauge Oxidative Stress in Breast Cancer Models.

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.