Discovery of lirafugratinib (RLY-4008), a highly selective irreversible small-molecule inhibitor of FGFR2.

Fibroblast growth factor receptor (FGFR) kinase inhibitors have been shown to be effective in the treatment of intrahepatic cholangiocarcinoma and other advanced solid tumors harboring FGFR2 alterations, but the toxicity of these drugs frequently leads to dose reduction or interruption of treatment such that maximum efficacy cannot be achieved. The most common adverse effects are hyperphosphatemia caused by FGFR1 inhibition and diarrhea due to FGFR4 inhibition, as current therapies are not selective among the FGFRs. Designing selective inhibitors has proved difficult with conventional approaches because the orthosteric sites of FGFR family members are observed to be highly similar in X-ray structures. In this study, aided by analysis of protein dynamics, we designed a selective, covalent FGFR2 inhibitor. In a key initial step, analysis of long-timescale molecular dynamics simulations of the FGFR1 and FGFR2 kinase domains allowed us to identify differential motion in their P-loops, which are located adjacent to the orthosteric site. Using this insight, we were able to design orthosteric binders that selectively and covalently engage the P-loop of FGFR2. Our drug discovery efforts culminated in the development of lirafugratinib (RLY-4008), a covalent inhibitor of FGFR2 that shows substantial selectivity over FGFR1 (~250-fold) and FGFR4 (~5,000-fold) in vitro, causes tumor regression in multiple FGFR2-altered human xenograft models, and was recently demonstrated to be efficacious in the clinic at doses that do not induce clinically significant hyperphosphatemia or diarrhea.

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

RLY-4008, the First Highly Selective FGFR2 Inhibitor with Activity across FGFR2 Alterations and Resistance Mutations.

Oncogenic activation of fibroblast growth factor receptor 2 (FGFR2) drives multiple cancers and represents a broad therapeutic opportunity, yet selective targeting of FGFR2 has not been achieved. Although the clinical efficacy of pan-FGFR inhibitors (pan-FGFRi) validates FGFR2 driver status in FGFR2 fusion-positive intrahepatic cholangiocarcinoma, their benefit is limited by incomplete target coverage due to FGFR1- and FGFR4-mediated toxicities (hyperphosphatemia and diarrhea, respectively) and the emergence of FGFR2 resistance mutations. RLY-4008 is a highly selective, irreversible FGFR2 inhibitor designed to overcome these limitations. In vitro, RLY-4008 demonstrates >250- and >5,000-fold selectivity over FGFR1 and FGFR4, respectively, and targets primary alterations and resistance mutations. In vivo, RLY-4008 induces regression in multiple xenograft models-including models with FGFR2 resistance mutations that drive clinical progression on current pan-FGFRi-while sparing FGFR1 and FGFR4. In early clinical testing, RLY-4008 induced responses without clinically significant off-isoform FGFR toxicities, confirming the broad therapeutic potential of selective FGFR2 targeting. SIGNIFICANCE: Patients with FGFR2-driven cancers derive limited benefit from pan-FGFRi due to multiple FGFR1-4-mediated toxicities and acquired FGFR2 resistance mutations. RLY-4008 is a highly selective FGFR2 inhibitor that targets primary alterations and resistance mutations and induces tumor regression while sparing other FGFRs, suggesting it may have broad therapeutic potential. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.

Computational fluid dynamic analysis of upper airway procedures in equine larynges.

Introduction: Computational fluid dynamics (CFD) has proven useful in the planning of upper airway surgery in humans, where it is used to anticipate the influence of the surgical procedures on post-operative airflow. This technology has only been reported twice in an equine model, with a limited scope of airflow mechanics situations examined. The reported study sought to widen this application to the variety of procedures used to treat equine recurrent laryngeal neuropathy (RLN). The first objective of this study was to generate a CFD model of an ex-vivo box model of ten different equine larynges replicating RLN and four therapeutic surgeries to compare the calculated impedance between these procedures for each larynx. The second objective was to determine the accuracy between a CFD model and measured airflow characteristics in equine larynges. The last objective was to explore the anatomic distribution of changes in pressure, velocity, and turbulent kinetic energy associated with the disease (RLN) and each surgical procedure performed. Methods: Ten equine cadaveric larynges underwent inhalation airflow testing in an instrumented box while undergoing a concurrent computed tomographic (CT) exam. The pressure upstream and downstream (outlet) were measured simultaneously. CT image segmentation was performed to generate stereolithography files, which underwent CFD analysis using the experimentally measured outlet pressure. The ranked procedural order and calculated laryngeal impedance were compared to the experimentally obtained values. Results and discussion: The CFD model agreed with the measured results in predicting the procedure resulting in the lowest post-operative impedance in 9/10 larynges. Numerically, the CFD calculated laryngeal impedance was approximately 0.7 times that of the measured calculation. Low pressure and high velocity were observed around regions of tissue protrusion within the lumen of the larynx. RLN, the corniculectomy and partial arytenoidectomy surgical procedures exhibited low pressure troughs and high velocity peaks compared to the laryngoplasty and combined laryngoplasty/corniculectomy procedures. CFD modeling of the equine larynx reliably calculated the lowest impedance of the different surgical procedures. Future development of the CFD technique to this application may improve numerical accuracy and is recommended prior to consideration for use in patients.

Dolaflexin: A Novel Antibody-Drug Conjugate Platform Featuring High Drug Loading and a Controlled Bystander Effect.

After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

The Dolaflexin-based Antibody-Drug Conjugate XMT-1536 Targets the Solid Tumor Lineage Antigen SLC34A2/NaPi2b.

Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical in vitro and in vivo antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.

Construction and optimization of gene expression signatures for prediction of survival in two-arm clinical trials.

BACKGROUND: Gene expression signatures for the prediction of differential survival of patients undergoing anti-cancer therapies are of great interest because they can be used to prospectively stratify patients entering new clinical trials, or to determine optimal treatment for patients in more routine clinical settings. Unlike prognostic signatures however, predictive signatures require training set data from clinical studies with at least two treatment arms. As two-arm studies with gene expression profiling have been rarer than similar one-arm studies, the methodology for constructing and optimizing predictive signatures has been less prominently explored than for prognostic signatures. RESULTS: Focusing on two "use cases" of two-arm clinical trials, one for metastatic colorectal cancer (CRC) patients treated with the anti-angiogenic molecule aflibercept, and the other for triple negative breast cancer (TNBC) patients treated with the small molecule iniparib, we present derivation steps and quantitative and graphical tools for the construction and optimization of signatures for the prediction of progression-free survival based on cross-validated multivariate Cox models. This general methodology is organized around two more specific approaches which we have called subtype correlation (subC) and mechanism-of-action (MOA) modeling, each of which leverage a priori knowledge of molecular subtypes of tumors or drug MOA for a given indication. The tools and concepts presented here include the so-called differential log-hazard ratio, the survival scatter plot, the hazard ratio receiver operating characteristic, the area between curves and the patient selection matrix. In the CRC use case for instance, the resulting signature stratifies the patient population into "sensitive" and "relatively-resistant" groups achieving a more than two-fold difference in the aflibercept-to-control hazard ratios across signature-defined patient groups. Through cross-validation and resampling the probability of generalization of the signature to similar CRC data sets is predicted to be high. CONCLUSIONS: The tools presented here should be of general use for building and using predictive multivariate signatures in oncology and in other therapeutic areas.

Novel, Self-Assembling Dimeric Inhibitors of Human ß Tryptase.

ß-Tryptase, a homotetrameric serine protease, has four identical active sites facing a central pore, presenting an optimized setting for the rational design of bivalent inhibitors that bridge two adjacent sites. Using diol, hydroxymethyl phenols or benzoyl methyl hydroxamates, and boronic acid chemistries to reversibly join two [3-(1-acylpiperidin-4-yl)phenyl]methanamine core ligands, we have successfully produced a series of self-assembling heterodimeric inhibitors. These heterodimeric tryptase inhibitors demonstrate superior activity compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and compounds demonstrated high selectivity against related proteases, good target engagement, and tryptase inhibition in HMC1 xenograft models. Screening 3872 possible combinations from 44 boronic acid and 88 diol derivatives revealed several combinations that produced nanomolar inhibition, and seven unique pairs produced greater than 100-fold improvement in potency over monomeric inhibition. These heterodimeric tryptase inhibitors demonstrate the power of target-driven combinatorial chemistry to deliver bivalent drugs in a small molecule form.

Target-Directed Self-Assembly of Homodimeric Drugs Against ß-Tryptase.

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC50 (0.19 ± 0.08 µM) over controls (5.50 ± 0.09 µM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets.

A Novel, Nonpeptidic, Orally Active Bivalent Inhibitor of Human ß-Tryptase.

ß-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.

Reversible linkage of two distinct small molecule inhibitors of Myc generates a dimeric inhibitor with improved potency that is active in myc over-expressing cancer cell lines.

We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

Evaluating patient-derived colorectal cancer xenografts as preclinical models by comparison with patient clinical data.

Development of targeted therapeutics required translationally relevant preclinical models with well-characterized cancer genome alterations. Here, by studying 52 colorectal patient-derived tumor xenografts (PDX), we examined key molecular alterations of the IGF2-PI3K and ERBB-RAS pathways and response to cetuximab. PDX molecular data were compared with that published for patient colorectal tumors in The Cancer Genome Atlas. We demonstrated a significant pattern of mutual exclusivity of genomic abnormalities in the IGF2-PI3K and ERBB-RAS pathways. The genomic anomaly frequencies observed in microsatellite stable PDX reproduce those detected in nonhypermutated patient tumors. We found frequent IGF2 upregulation (16%), which was mutually exclusive with IRS2, PIK3CA, PTEN, and INPP4B alterations, supporting IGF2 as a potential drug target. In addition to maintaining the genomic and histologic diversity, correct preclinical models need to reproduce drug response observed in patients. Responses of PDXs to cetuximab recapitulate also clinical data in patients, with partial or complete response in 15% (8 of 52) of PDXs and response strictly restricted to KRAS wild-type models. The response rate reaches 53% (8 of 15) when KRAS, BRAF, and NRAS mutations are concomitantly excluded, proving a functional cross-validation of predictive biomarkers obtained retrospectively in patients. Collectively, these results show that, because of their clinical relevance, colorectal PDXs are appropriate tools to identify both new targets, like IGF2, and predictive biomarkers of response/resistance to targeted therapies.

Differential antitumor activity of aflibercept and bevacizumab in patient-derived xenograft models of colorectal cancer.

The recombinant fusion protein aflibercept (ziv-aflibercept in the United States) binds VEGF-A, VEGF-B, and placental growth factor (PlGF). The monoclonal antibody bevacizumab binds VEGF-A. Recent studies hypothesized that dual targeting of VEGF/PlGF is more beneficial than targeting either ligand. We compared activity of aflibercept versus bevacizumab in 48 patient-derived xenograft (PDX) colorectal cancer models. Nude mice engrafted subcutaneously with PDX colorectal cancer tumors received biweekly aflibercept, bevacizumab, or vehicle injections. Differential activity between aflibercept and bevacizumab, determined by mouse (m), human (h), VEGF-A, and PlGF levels in untreated tumors, was measured. Aflibercept induced complete tumor stasis in 31 of 48 models and bevacizumab in 2 of 48. Based on statistical analysis, aflibercept was more active than bevacizumab in 39 of 48 models; in 9 of 39 of these models, bevacizumab was considered inactive. In 9 of 48 remaining models, aflibercept and bevacizumab had similar activity. Tumor levels of hVEGF-A (range 776-56,039 pg/mg total protein) were ∼16- to 1,777-fold greater than mVEGF-A (range 8-159 pg/mg total protein). Tumor levels of mPlGF (range 104-1,837 pg/mg total protein) were higher than hPlGF (range 0-543 pg/mg total protein) in 47 of 48 models. Tumor cells were the major source of VEGF; PlGF was primarily produced by tumor stroma. Because tumor levels of hVEGF-A were far greater than mVEGF-A, bevacizumab's inability to bind mVEGF-A is unlikely to explain higher and more consistent aflibercept activity. Neutralizing PlGF and VEGFR-1 activation may be a factor and should be investigated in future studies. In these colorectal cancer PDX models, aflibercept demonstrated greater antitumor activity than bevacizumab.

Repeatability of quantitative FDG-PET/CT and contrast-enhanced CT in recurrent ovarian carcinoma: test-retest measurements for tumor FDG uptake, diameter, and volume.

PURPOSE: Repeatability of baseline FDG-PET/CT measurements has not been tested in ovarian cancer. This dual-center, prospective study assessed variation in tumor 2[18F]fluoro-2-deoxy-D-glucose (FDG) uptake, tumor diameter, and tumor volume from sequential FDG-PET/CT and contrast-enhanced computed tomography (CECT) in patients with recurrent platinum-sensitive ovarian cancer. EXPERIMENTAL DESIGN: Patients underwent two pretreatment baseline FDG-PET/CT (n = 21) and CECT (n = 20) at two clinical sites with different PET/CT instruments. Patients were included if they had at least one target lesion in the abdomen with a standardized uptake value (SUV) maximum (SUVmax) of ≥ 2.5 and a long axis diameter of ≥ 15 mm. Two independent reading methods were used to evaluate repeatability of tumor diameter and SUV uptake: on site and at an imaging clinical research organization (CRO). Tumor volume reads were only performed by CRO. In each reading set, target lesions were independently measured on sequential imaging. RESULTS: Median time between FDG-PET/CT was two days (range 1-7). For site reads, concordance correlation coefficients (CCC) for SUVmean, SUVmax, and tumor diameter were 0.95, 0.94, and 0.99, respectively. Repeatability coefficients were 16.3%, 17.3%, and 8.8% for SUVmean, SUVmax, and tumor diameter, respectively. Similar results were observed for CRO reads. Tumor volume CCC was 0.99 with a repeatability coefficient of 28.1%. CONCLUSIONS: There was excellent test-retest repeatability for FDG-PET/CT quantitative measurements across two sites and two independent reading methods. Cutoff values for determining change in SUVmean, SUVmax, and tumor volume establish limits to determine metabolic and/or volumetric response to treatment in platinum-sensitive relapsed ovarian cancer.

4-Aminocyclopentane-1,3-diols as platforms for diversity: synthesis of anandamide analogs.

Starting from cyclopentadiene, two racemic mixtures of 4-aminocyclopentane-1,3-diols were prepared in 8 steps and characterized. Structure determination proved the anticipated trans-orientation of the two oxygen atoms with respect to the plane of the ring. The fragment-like new compounds are small and hydrophilic, devoid of rotatable bonds, and offer stereochemically defined attachment points for substituents. Thus, these platforms for diversity are suitable starting points for the construction of combinatorial libraries of lead-like 4-amidocyclopentane-1,3-diols or natural product analogs. As a proof of concept, cyclopentanoid anandamide analogs were prepared using these molecular platforms and evaluated as tools for the investigation of unresolved issues in the molecular biology of anandamide.

1,2,4-trisubstituted cyclopentanes as platforms for diversity.

Despite their simplicity, relatively few examples of 1,2,4 (1,3,4)-amino-, azido-, and hydroxy-substituted cyclopentanes are reported in the literature. We found that cyclopent-3-en-1-ol can be transformed into a significant variety of compounds of this class by relatively common and efficient synthetic procedures. Stereochemical control of epoxidation of the cyclopentene double bond can be achieved by varying the substitutents at C4. The C4 substituent and epoxide functional group can be converted into a variety of intermediates with differential protection designed for use in applications requiring regiospecific control for further elaboration of the cyclopentane scaffold.

Evaluation of end-capped DNA modules for pRNA capture and functionalization.

The ability of packaging RNA (pRNA) from the phi29 DNA packaging motor to form nanoassemblies and nanostructures has been exploited for the development of the nascent field of RNA nanotechnology and subsequent applications in nanomedicine. For applications in nanomedicine, it is necessary to modify the pRNA structure for the conjugation of active molecules. We have investigated end-capped double-stranded DNA segments as reversible capture reagents for pRNA. These capture agents can be designed to allow the conjugation of any desired molecule for pRNA functionalization. The results of model studies presented in this report show that 5- to 7-nucleotide overhangs on a target RNA can provide efficient handles for the high-affinity association to capped double-stranded DNA.

Label-free imaging of semiconducting and metallic carbon nanotubes in cells and mice using transient absorption microscopy.

As interest in the potential biomedical applications of carbon nanotubes increases, there is a need for methods that can image nanotubes in live cells, tissues and animals. Although techniques such as Raman, photoacoustic and near-infrared photoluminescence imaging have been used to visualize nanotubes in biological environments, these techniques are limited because nanotubes provide only weak photoluminescence and low Raman scattering and it remains difficult to image both semiconducting and metallic nanotubes at the same time. Here, we show that transient absorption microscopy offers a label-free method to image both semiconducting and metallic single-walled carbon nanotubes in vitro and in vivo, in real time, with submicrometre resolution. By using appropriate near-infrared excitation wavelengths, we detect strong transient absorption signals with opposite phases from semiconducting and metallic nanotubes. Our method separates background signals generated by red blood cells and this allows us to follow the movement of both types of nanotubes inside cells and in the blood circulation and organs of mice without any significant damaging effects.

Silicon field effect transistors as dual-use sensor-heater hybrids.

We demonstrate the temperature mediated applications of a previously proposed novel localized dielectric heating method on the surface of dual purpose silicon field effect transistor (FET) sensor-heaters and perform modeling and characterization of the underlying mechanisms. The FETs are first shown to operate as electrical sensors via sensitivity to changes in pH in ionic fluids. The same devices are then demonstrated as highly localized heaters via investigation of experimental heating profiles and comparison to simulation results. These results offer further insight into the heating mechanism and help determine the spatial resolution of the technique. Two important biosensor platform applications spanning different temperature ranges are then demonstrated: a localized heat-mediated DNA exchange reaction and a method for dense selective functionalization of probe molecules via the heat catalyzed complete desorption and reattachment of chemical functionalization to the transistor surfaces. Our results show that the use of silicon transistors can be extended beyond electrical switching and field-effect sensing to performing localized temperature controlled chemical reactions on the transistor itself.