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.

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.

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.

Intrastrand annealing leads to the formation of a large DNA palindrome and determines the boundaries of genomic amplification in human cancer.

Amplification of large chromosomal regions (gene amplification) is a common somatic alteration in human cancer cells and often is associated with advanced disease. A critical event initiating gene amplification is a DNA double-strand break (DSB), which is immediately followed by the formation of a large DNA palindrome. Large DNA palindromes are frequent and nonrandomly distributed in the genomes of cancer cells and facilitate a further increase in copy number. Although the importance of the formation of large DNA palindromes as a very early event in gene amplification is widely recognized, it is not known how a DSB is resolved to form a large DNA palindrome and whether any local DNA structure determines the location of large DNA palindromes. We show here that intrastrand annealing following a DNA double-strand break leads to the formation of large DNA palindromes and that DNA inverted repeats in the genome determine the efficiency of this event. Furthermore, in human Colo320DM cancer cells, a DNA inverted repeat in the genome marks the border between amplified and nonamplified DNA. Therefore, an early step of gene amplification is a regulated process that is facilitated by DNA inverted repeats in the genome.

Large DNA palindromes as a common form of structural chromosome aberrations in human cancers.

Breakage-fusion-bridge cycles contribute to chromosome aberrations and generate large DNA palindromes that facilitate oncogene amplification in cancer cells. At the molecular level, large DNA palindrome formation is initiated by chromosome breaks, and genomic architecture such as short inverted repeat sequences facilitates this process in mammalian cells. However, the prevalence of DNA palindromes in cancer cells is currently unknown. To determine the prevalence of DNA palindromes in human cancer cells, we have developed a new microarray-based approach called Genome-wide Analysis of Palindrome Formation (GAPF, Tanaka et al., Nat Genet 2005; 37: 320-7). This approach is based on a relatively simple and efficient method to purify "snap-back DNA" from large DNA palindromes by intramolecular base-pairing, followed by elimination of single-stranded DNA by nuclease S1. Comparison of Genome-wide Analysis of Palindrome Formation profiles between cancer and normal cells using microarray can identify genome-wide distributions of somatic palindromes. Using a human cDNA microarray, we have shown that DNA palindromes occur frequently in human cancer cell lines and primary medulloblastomas. Significant overlap of the loci containing DNA palindromes between Colo320DM and MCF7 cancer cell lines suggests regions in the genome susceptible to chromosome breaks and palindrome formation. A subset of loci containing palindromes is associated with gene amplification in Colo320DM, indicating that the location of palindromes in the cancer genome serves as a structural platform that supports subsequent gene amplification.

MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.

The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins.

Widespread and nonrandom distribution of DNA palindromes in cancer cells provides a structural platform for subsequent gene amplification.

Breakage-fusion-bridge cycles contribute to chromosome instability and generate large DNA palindromes that facilitate gene amplification in human cancers. The prevalence of large DNA palindromes in cancer is not known. Here, by using a new microarray-based approach called genome-wide analysis of palindrome formation, we show that palindromes occur frequently and are widespread in human cancers. Individual tumors seem to have a nonrandom distribution of palindromes in their genomes, and a subset of palindromic loci is associated with gene amplification. This indicates that the location of palindromes in the cancer genome can serve as a structural platform that supports subsequent gene amplification. Genome-wide analysis of palindrome formation is a new approach to identify structural chromosome aberrations associated with cancer.

A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation.

The development and differentiation of distinct cell types is achieved through the sequential expression of subsets of genes; yet, the molecular mechanisms that temporally pattern gene expression remain largely unknown. In skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Here, we show that p38 activity facilitates MyoD and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits Pol II. Most importantly, expression of late-activated genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that a MyoD-mediated feed-forward circuit temporally patterns gene expression.

Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential.

Skeletal muscle differentiation is initiated by the transcription factor MyoD, which binds directly to the regulatory regions of genes expressed during skeletal muscle differentiation and initiates chromatin remodeling at specific promoters. It is not known, however, how MyoD initially recognizes its binding site in a chromatin context. Here we show that the H/C and helix III domains, two domains of MyoD that are necessary for the initiation of chromatin remodeling at the myogenin locus, together regulate a restricted subset of genes, including myogenin. These domains are necessary for the stable binding of MyoD to the myogenin promoter through an interaction with an adjacent protein complex containing the homeodomain protein Pbx, which appears to be constitutively bound at this site. This demonstrates a specific mechanism of targeting MyoD to loci in inactive chromatin and reveals a critical role of homeodomain proteins in marking specific genes for activation in the muscle lineage.

Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain.

Previous analyses of gene expression in a mouse model of Huntington's disease (R6/2) indicated that an N-terminal fragment of mutant huntingtin causes downregulation of striatal signaling genes and particularly those normally induced by cAMP and retinoic acid. The present study expands the regional and temporal scope of this previous work by assessing whether similar changes occur in other brain regions affected in Huntington's disease and other polyglutamine diseases and by discerning whether gene expression changes precede the appearance of disease signs. Oligonucleotide microarrays were employed to survey the expression of approximately 11,000 mRNAs in the cerebral cortex, cerebellum and striatum of symptomatic R6/2 mice. The number and nature of gene expression changes were similar among these three regions, influenced as expected by regional differences in baseline gene expression. Time-course studies revealed that mRNA changes could only reliably be detected after 4 weeks of age, coincident with development of early pathologic and behavioral changes in these animals. In addition, we discovered that skeletal muscle is also a target of polyglutamine-related perturbations in gene expression, showing changes in mRNAs that are dysregulated in brain and also muscle-specific mRNAs. The complete dataset is available at www.neumetrix.info.

Promoter-specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression.

We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.