Targeted protein degradation: from mechanisms to clinic.

Targeted protein degradation refers to the use of small molecules to induce the selective degradation of proteins. In its most common form, this degradation is achieved through ligand-mediated neo-interactions between ubiquitin E3 ligases - the principal waste disposal machines of a cell - and the protein targets of interest, resulting in ubiquitylation and subsequent proteasomal degradation. Notable advances have been made in biological and mechanistic understanding of serendipitously discovered degraders. This improved understanding and novel chemistry has not only provided clinical proof of concept for targeted protein degradation but has also led to rapid growth of the field, with dozens of investigational drugs in active clinical trials. Two distinct classes of protein degradation therapeutics are being widely explored: bifunctional PROTACs and molecular glue degraders, both of which have their unique advantages and challenges. Here, we review the current landscape of targeted protein degradation approaches and how they have parallels in biological processes. We also outline the ongoing clinical exploration of novel degraders and provide some perspectives on the directions the field might take.

Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.

Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability.

Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.

Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy.

Exciting progress in the field of cancer immunotherapy has renewed the urgency of the need for basic studies of immunoregulation in both adaptive cell lineages and innate cell lineages. Here we found a central role for major histocompatibility complex (MHC) class I in controlling the phagocytic function of macrophages. Our results demonstrated that expression of the common MHC class I component ß2-microglobulin (ß2M) by cancer cells directly protected them from phagocytosis. We further showed that this protection was mediated by the inhibitory receptor LILRB1, whose expression was upregulated on the surface of macrophages, including tumor-associated macrophages. Disruption of either MHC class I or LILRB1 potentiated phagocytosis of tumor cells both in vitro and in vivo, which defines the MHC class I-LILRB1 signaling axis as an important regulator of the effector function of innate immune cells, a potential biomarker for therapeutic response to agents directed against the signal-regulatory protein CD47 and a potential target of anti-cancer immunotherapy.

Template-assisted covalent modification underlies activity of covalent molecular glues.

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.

Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

Complex mammalian-like haematopoietic system found in a colonial chordate.

Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life1. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin's lymphoma. Although it is well established that PD-1-PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1-PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1-PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.

MET D1228N and D1246N are the Same Resistance Mutation in MET Exon 14 Skipping.

Comprehensive genetic profiling using next-generation sequencing technologies has become an integral part of precision oncology. Variant annotation requires translating the DNA findings into protein level predictions. In this article we highlight inconsistencies in variant annotation for the MET D1228N exon 19 resistance mutations. MET D1228N and D1246N represent the same resistance mutation in MET exon 14 skipping alterations annotated on different transcripts. Additional examples of relevant variants annotated on different transcripts emphasize the importance of avoiding erroneous interpretation when realizing precision oncology.

Indolent CD8+ primary cutaneous T-cell lymphoma involving the eyelid of an adolescent.

Primary cutaneous acral CD8+ T-cell lymphoma (PCACTL) is currently a provisional entity defined as a rare cutaneous proliferation of atypical CD8+ lymphocytes that preferentially involves acral sites and has a good prognosis. We present a case of primary cutaneous CD8+ T-cell lymphoma involving the eyelid of an adolescent male. The case shares features with PCACTL, including indolent clinical behavior and expression of CD68 in a Golgi-associated dot-like pattern; however, other features differ significantly from PCACTL as currently defined by the World Health Organization (WHO). These features include ulceration, expression of CD56, granzyme B, and perforin, and a high proliferative index. Given these discrepancies, our case is currently best classified as a CD8+ primary cutaneous peripheral T-cell lymphoma, not otherwise specified. We review the differential diagnosis for this case and suggest expanding the definition of PCACTL.

Wnt/ß-catenin signaling regulates ependymal cell development and adult homeostasis.

In the adult mouse spinal cord, the ependymal cell population that surrounds the central canal is thought to be a promising source of quiescent stem cells to treat spinal cord injury. Relatively little is known about the cellular origin of ependymal cells during spinal cord development, or the molecular mechanisms that regulate ependymal cells during adult homeostasis. Using genetic lineage tracing based on the Wnt target gene Axin2, we have characterized Wnt-responsive cells during spinal cord development. Our results revealed that Wnt-responsive progenitor cells are restricted to the dorsal midline throughout spinal cord development, which gives rise to dorsal ependymal cells in a spatially restricted pattern. This is contrary to previous reports that suggested an exclusively ventral origin of ependymal cells, suggesting that ependymal cells may retain positional identities in relation to their neural progenitors. Our results further demonstrated that in the postnatal and adult spinal cord, all ependymal cells express the Wnt/ß-catenin signaling target gene Axin2, as well as Wnt ligands. Genetic elimination of ß-catenin or inhibition of Wnt secretion in Axin2-expressing ependymal cells in vivo both resulted in impaired proliferation, indicating that Wnt/ß-catenin signaling promotes ependymal cell proliferation. These results demonstrate the continued importance of Wnt/ß-catenin signaling for both ependymal cell formation and regulation. By uncovering the molecular signals underlying the formation and regulation of spinal cord ependymal cells, our findings thus enable further targeting and manipulation of this promising source of quiescent stem cells for therapeutic interventions.

Localized hepatic lobular regeneration by central-vein-associated lineage-restricted progenitors.

The regeneration of organ morphology and function following tissue loss is critical to restore normal physiology, yet few cases are documented in mammalian postnatal life. Partial hepatectomy of the adult mammalian liver activates compensatory hepatocyte hypertrophy and cell division across remaining lobes, resulting in restitution of organ mass but with permanent alteration of architecture. Here, we identify a time window in early postnatal life wherein partial amputation culminates in a localized regeneration instead of global hypertrophy and proliferation. Quantifications of liver mass, enzymatic activity, and immunohistochemistry demonstrate that damaged lobes underwent multilineage regeneration, reforming a lobe often indistinguishable from undamaged ones. Clonal analysis during regeneration reveals local clonal expansions of hepatocyte stem/progenitors at injured sites that are lineage but not fate restricted. Tetrachimeric mice show clonal selection occurs during development with further selections following injury. Surviving progenitors associate mainly with central veins, in a pattern of selection different from that of normal development. These results illuminate a previously unknown program of liver regeneration after acute injury and allow for exploration of latent regenerative programs with potential applications to adult liver regeneration.

Unifying mechanism for different fibrotic diseases.

Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun-mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

Coral cell separation and isolation by fluorescence-activated cell sorting (FACS).

BACKGROUND: Generalized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians. METHODS: Using fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay. RESULTS: We found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker. CONCLUSIONS: In this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies.

The Incidence of Ocular Injuries in Isolated Orbital Fractures.

BACKGROUND: Prompt identification of significant ocular injuries in patients who sustain an orbital fracture is important to prevent any potential long-term visual sequelae. The true incidence of these injuries has not been determined, however. As a consequence, most surgeons choose to have all patients evaluated by an ophthalmologist. The objective of this study was to conclusively identify the incidence of significant ocular injuries in patients with isolated orbital fractures and to determine their predictors to guide more efficient patient care. METHODS: A prospective cohort study powered to detect a 15% incidence of ocular injuries was designed. All patients presenting to our center with computed tomography findings of an isolated orbital fracture were included and evaluated by plastic surgery and ophthalmology services. Patients were followed up for a minimum of 1 week to identify any delayed injuries. RESULTS: Eighty patients were enrolled from 2012 to 2014. There were 46 men and 34 women with a mean age of 42.8 years. Assault was the most common mechanism of injury. There were 8 ocular injuries (10%): ruptured globe (1), uveal prolapse (1), retrobulbar hemorrhage (2), hyphema (2), hemorrhagic glaucoma with hyphema (1), and scleral tear (1). Predictors for significant ocular injuries were grossly abnormal visual acuity and abnormal pupillary reactivity of the affected eye. CONCLUSIONS: The incidence of significant ocular injuries in isolated orbital fractures is lower than previously reported. Patients presenting with grossly abnormal visual acuity or abnormal pupillary reactivity are at high risk and should receive prompt ophthalmology service evaluation.

The human E3 ligase RNF185 is a regulator of the SARS-CoV-2 envelope protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks multiple human proteins during infection and viral replication. To examine whether any viral proteins employ human E3 ubiquitin ligases, we evaluated the stability of SARS-CoV-2 proteins with inhibition of the ubiquitin proteasome pathway. Using genetic screens to dissect the molecular machinery involved in the degradation of candidate viral proteins, we identified human E3 ligase RNF185 as a regulator of protein stability for the SARS-CoV-2 envelope protein. We found that RNF185 and the SARS-CoV-2 envelope co-localize to the endoplasmic reticulum (ER). Finally, we demonstrate that the depletion of RNF185 significantly increases SARS-CoV-2 viral titer in a cellular model. Modulation of this interaction could provide opportunities for novel antiviral therapies.

Outlier Expression of Isoforms by Targeted or Total RNA Sequencing Identifies Clinically Significant Genomic Variants in Hematolymphoid Tumors.

Recognition of aberrant gene isoforms due to DNA events can impact risk stratification and molecular classification of hematolymphoid tumors. In myelodysplastic syndromes, KMT2A partial tandem duplication (PTD) was one of the top adverse predictors in the International Prognostic Scoring System-Molecular study. In B-cell acute lymphoblastic leukemia (B-ALL), ERG isoforms have been proposed as markers of favorable-risk DUX4 rearrangements, whereas deletion-mediated IKZF1 isoforms are associated with adverse prognosis and have been extended to the high-risk IKZF1plus signature defined by codeletions, including PAX5. In this limited study, outlier expression of isoforms as markers of IKZF1 intragenic or 3' deletions, DUX4 rearrangements, or PAX5 intragenic deletions were 92.3% (48/52), 90% (9/10), or 100% (9/9) sensitive, respectively, and 98.7% (368/373), 100% (35/35), or 97.1% (102/105) specific, respectively, by targeted RNA sequencing, and 84.0% (21/25), 85.7% (6/7), or 81.8% (9/11) sensitive, respectively, and 98.2% (109/111), 98.4% (127/129), or 98.7% (78/79) specific, respectively, by total RNA sequencing. Comprehensive split-read analysis identified expressed DNA breakpoints, cryptic splice sites associated with IKZF1 3' deletions, PTD of IKZF1 exon 5 spanning N159Y in B-ALL with mutated IKZF1 N159Y, and truncated KMT2A-PTD isoforms. Outlier isoforms were also effective targeted RNA markers for PAX5 intragenic amplifications (B-ALL), KMT2A-PTD (myeloid malignant cancers), and rare NOTCH1 intragenic deletions (T-cell acute lymphoblastic leukemia). These findings support the use of outlier isoform analysis as a robust strategy for detecting clinically significant DNA events.

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders.

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

Integration of Genomic Sequencing Drives Therapeutic Targeting of PDGFRA in T-Cell Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma.

PURPOSE: Patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) or lymphoblastic lymphoma (T-LBL) have limited therapeutic options. Clinical use of genomic profiling provides an opportunity to identify targetable alterations to inform therapy. EXPERIMENTAL DESIGN: We describe a cohort of 14 pediatric patients with relapsed or refractory T-ALL enrolled on the Leukemia Precision-based Therapy (LEAP) Consortium trial (NCT02670525) and a patient with T-LBL, discovering alterations in platelet-derived growth factor receptor-α (PDGFRA) in 3 of these patients. We identified a novel mutation in PDGFRA, p.D842N, and used an integrated structural modeling and molecular biology approach to characterize mutations at D842 to guide therapeutic targeting. We conducted a preclinical study of avapritinib in a mouse patient-derived xenograft (PDX) model of FIP1L1-PDGFRA and PDGFRA p.D842N leukemia. RESULTS: Two patients with T-ALL in the LEAP cohort (14%) had targetable genomic alterations affecting PDGFRA, a FIP1-like 1 protein/PDGFRA (FIP1L1-PDGFRA) fusion and a novel mutation in PDGFRA, p.D842N. The D842N mutation resulted in PDGFRA activation and sensitivity to tested PDGFRA inhibitors. In a T-ALL PDX model, avapritinib treatment led to decreased leukemia burden, significantly prolonged survival, and even cured a subset of mice. Avapritinib treatment was well tolerated and yielded clinical benefit in a patient with refractory T-ALL. CONCLUSIONS: Refractory T-ALL has not been fully characterized. Alterations in PDGFRA or other targetable kinases may inform therapy for patients with refractory T-ALL who otherwise have limited treatment options. Clinical genomic profiling, in real time, is needed for fully informed therapeutic decision making.