NRX-0492 degrades wild-type and C481 mutant BTK and demonstrates in vivo activity in CLL patient-derived xenografts.

Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).

PI3K inhibitors in chronic lymphocytic leukemia: where do we go from here?

Phosphatidylinositol 3-kinase (PI3K) inhibitors are effective in chronic lymphocytic leukemia (CLL). However, the severe toxicity profile associated with the first-generation inhibitors idelalisib and duvelisib, combined with the availability of other more tolerable agents, have limited their use. CLL is still considered incurable, and relapse after treatment, development of resistance, and treatment intolerance are common. It is therefore of interest to optimize the administration of currently approved PI3K inhibitors and to develop next-generation agents to improve tolerability, so that this class of agents will be considered an effective and safe treatment option when needed. These efforts are reflected in the large number of emerging clinical trials with PI3K inhibitors in CLL. Current strategies to overcome treatment limitations include intermittent dosing, which is established for copanlisib and zandelisib and under investigation for duvelisib and parsaclisib. A second strategy is to combine the PI3K inhibitor with another novel agent, either as a continuous regimen or a fixedduration regimen, to deepen responses. In addition to these approaches, it is of interest to identify higher-resolution actionable biomarkers that can predict treatment responses and toxicity, and inform personalized treatment decisions. Here, we discuss the current status of PI3K inhibitors in CLL, factors limiting the use of currently approved PI3K inhibitors in CLL, current strategies to overcome these limitations, and where to go next.

T-helper cell regulation of CD45 phosphatase activity by galectin-1 and CD43 governs chronic lymphocytic leukaemia proliferation.

Chronic lymphocytic leukaemia (CLL) is characterised by malignant mature-like B cells. Supportive to CLL cell survival is chronic B-cell receptor (BCR) signalling; however, emerging evidence demonstrates CLL cells proliferate in response to T-helper (Th) cells in a CD40L-dependent manner. We showed provision of Th stimulation via CD40L upregulated CD45 phosphatase activity and BCR signalling in non-malignant B cells. Consequently, we hypothesised Th cell upregulation of CLL cell CD45 activity may be an important regulator of CLL BCR signalling and proliferation. Using patient-derived CLL cells in a culture system with activated autologous Th cells, results revealed increases in both Th and CLL cell CD45 activity, which correlated with enhanced downstream antigen receptor signalling and proliferation. Concomitantly increased was the surface expression of Galectin-1, a CD45 ligand, and CD43, a CLL immunophenotypic marker. Galectin-1/CD43 double expression defined a proliferative CLL cell population with enhanced CD45 activity. Targeting either Galectin-1 or CD43 using silencing, pharmacology, or monoclonal antibody strategies dampened CD45 activity and CLL cell proliferation. These results highlight a mechanism where activated Th cells drive CLL cell BCR signalling and proliferation via Galectin-1 and CD43-mediated regulation of CD45 activity, identifying modulation of CD45 phosphatase activity as a potential therapeutic target in CLL.

Approach to a patient with "double refractory" chronic lymphocytic leukemia: "Double, double toil and trouble" (Shakespeare).

As patients continue to live longer with chronic lymphocytic leukemia, it has become evident that there is an unmet treatment need for patients who have progressed on multiple lines of therapy. In this article, we attempt to define the "double refractory" patient as resistant to both Bruton's tyrosine kinase inhibitors (BTKi) and venetoclax for which prognosis is poor and there remains no standard of care. We further examine the mechanism of resistance to these targeted agents and discuss the current landscape for managing this patient population. Finally, we explore data supporting promising new agents, including non-covalent BTKi, chimeric antigen receptor T cells, and additional classes of agents currently in development.

Carboxyl-Terminal Src Kinase Binds CD28 upon Activation and Mutes Downstream Signaling.

Full T cell activation depends on stimulation of the TCR in conjunction with a costimulatory receptor. The involvement of costimulatory molecules is potent, and a mechanistic understanding of how downstream signaling is regulated is required to fully understand T cell responsiveness. In this study, a proteomic approach was taken to identify the interactomes of the coreceptors CD2 and CD28. These coreceptors are both positive regulators of T cell activation, but CD28 less potently induces TCR-proximal signaling. C-terminal Src kinase (CSK), a negative regulator of TCR signaling, was identified as a specific and direct interactor only of activated CD28. CSK is recruited to CD28 upon T cell activation, and the in vitro kinase activity of CSK is enhanced in the presence of phosphorylated CD28. Interruption of the CSK/CD28 interaction prior to TCR/CD28 costimulation induces a signaling response which mimics the more potent CD2-induced TCR-proximal pathway activation. Thus, CD28 functions as a novel adaptor protein for CSK, and CSK regulates signaling downstream of CD28.

B cell signalling pathways-New targets for precision medicine in chronic lymphocytic leukaemia.

The B cell receptor (BCR) is a master regulator of B cells, controlling cellular processes such as proliferation, migration and survival. Cell signalling downstream of the BCR is aberrantly activated in the B cell malignancy chronic lymphocytic leukaemia (CLL), supporting the pathophysiology of the disease. This insight has led to development and approval of small molecule inhibitors that target components of the BCR pathway. These advances have greatly improved the management of CLL, but the disease remains incurable. This may partly be explained by the inter-patient heterogeneity of the disease, also when it comes to treatment responses. Precision medicine is therefore required to optimize treatment and move towards a cure. Here, we discuss how the introduction of BCR signalling inhibitors has facilitated the development of functional in vitro assays to guide clinical treatment decisions on use of the same therapeutic agents in individual patients. The cellular responses to these agents can be analysed in high-throughput assays such as dynamic BH3 profiling, phospho flow experiments and drug sensitivity screens to identify predictive biomarkers. This progress exemplifies the positive synergy between basal and translational research needed to optimize patient care.

SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis.

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

T-cell co-stimulation through the CD2 and CD28 co-receptors induces distinct signalling responses.

Full T-cell activation critically depends on the engagement of the TCR (T-cell receptor) in conjunction with a second signal by co-stimulatory receptors that boost the immune response. In the present study we have compared signalling patterns induced by the two co-receptors CD2 and CD28 in human peripheral blood T-cells. These co-receptors were previously suggested to be redundant in function. By a combination of multi-parameter phosphoflow cytometry, phosphokinase arrays and Western blot analyses, we demonstrate that CD2 co-stimulation induces phosphorylation of the TCR-proximal signalling complex, whereas CD28 activates distal signalling molecules, including the transcription factors NF-κB (nuclear factor κB), ATF (activating transcription factor)-2, STAT3/5 (signal transducer and activator of transcription 3/5), p53 and c-Jun. These signalling patterns were conserved in both naïve and effector/memory T-cell subsets. We show that free intracellular Ca(2+) and signalling through the PI3K (phosphoinositide 3-kinase)/Akt pathway are required for proper CD28-induced NF-κB activation. The signalling patterns induced by CD2 and CD28 co-stimulation lead to distinct functional immune responses in T-cell proliferation and cytokine production. In conclusion, CD2 and CD28 co-stimulation induces distinct signalling responses and functional outcomes in T-cells.

Spatial organization of transmembrane receptor signalling.

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis-interactions as well as intercellular trans-interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand-induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid-protein interactions important for the spatial organization of signalling at the membrane.

Interleukin-33 drives a proinflammatory endothelial activation that selectively targets nonquiescent cells.

OBJECTIVE: Interleukin (IL)-33 is a nuclear protein that is released from stressed or damaged cells to act as an alarmin. We investigated the effects of IL-33 on endothelial cells, using the prototype IL-1 family member, IL-1ß, as a reference. METHODS AND RESULTS: Human umbilical vein endothelial cells were stimulated with IL-33 or IL-1ß, showing highly similar phosphorylation of signaling molecules, induction of adhesion molecules, and transcription profiles. However, intradermally injected IL-33 elicited significantly less proinflammatory endothelial activation when compared with IL-1ß and led us to observe that quiescent endothelial cells (ppRb(low)p27(high)) were strikingly resistant to IL-33. Accordingly, the IL-33 receptor was preferentially expressed in nonquiescent cells of low-density cultures, corresponding to selective induction of adhesion molecules and chemokines. Multiparameter phosphoflow cytometry confirmed that signaling driven by IL-33 was stronger in nonquiescent cells. Manipulation of nuclear IL-33 expression by siRNA or adenoviral transduction revealed no functional link between nuclear, endogenous IL-33, and exogenous IL-33 responsiveness. CONCLUSIONS: In contrast to other inflammatory cytokines, IL-33 selectively targets nonquiescent endothelial cells. By this novel concept, quiescent cells may remain nonresponsive to a proinflammatory stimulus that concomitantly triggers a powerful response in cells that have been released from contact inhibition.

PI3K inhibitors in hematology: When one door closes .

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates key cellular processes and is one of the most aberrantly activated pathways in cancer. The class I PI3K catalytic subunits p110g and p110d are highly enriched in leukocytes, providing additional rationale for targeting these PI3Ks in hematologic malignancies. In 2014, the PI3Kd inhibitor idelalisib was the first of four PI3K inhibitors to receive regulatory approval for relapsed B-cell malignancies. This was followed by approvals of the pan-class I inhibitor copanlisib (2017), the dual PI3Kg/d inhibitor duvelisib (2018), and the PI3Kd and casein kinase-1e inhibitor umbralisib (2021). Copanlisib and umbralisib received accelerated approvals, while idelalisib and duvelisib received initial accelerated approvals followed by full approvals. The accelerated approvals were based on overall response rates, however follow-up studies showed increased risk of death and serious side effects. Furthermore, the confirmatory trial with copanlisib failed to show an improvement in progression free survival when compared to chemoimmunotherapy. These developments led to black box warnings for idelalisib and duvelisib, and withdrawal of copanlisib and umbralisib from the market by their manufacturers. Given the uncertain future of this drug class, additional manufacturers terminated ongoing phase III trials with novel PI3K inhibitors. Here, we review the development and current status of PI3K inhibitors in hematology, limitations to their use, and our perspective on whether there is a future for PI3K inhibitors in hematology.

Immunophenotyping with (phospho)protein profiling and fluorescent cell barcoding for single-cell signaling analysis and biomarker discovery.

The microenvironment of hematologic cancers contributes to tumor cell survival and proliferation, as well as treatment resistance. Understanding tumor- and drug-induced changes to the immune cell composition and functionality is therefore critical for implementing optimal treatment strategies and for the development of novel cancer therapies. The liquid nature of peripheral blood makes this organ uniquely suited for single-cell studies by flow cytometry. (Phospho)protein profiles detected by flow cytometry analyses have been shown to correlate with ex vivo drug sensitivity and to predict treatment outcomes in hematologic cancers, demonstrating that this method is suitable for pre-clinical studies. Here, we present a flow cytometry protocol that combines multi-parameter immunophenotyping with single-cell (phospho)protein profiling. The protocol makes use of fluorescent cell barcoding, which means that multiple cell samples, either collected from different donors or exposed to different treatment conditions, can be combined and analyzed as one experiment. This reduces variability between samples, increases the throughput of the experiment, and lowers experimental costs. This protocol may serve as a guide for the use and further development of assays to study immunophenotype and cell signaling at single-cell resolution in normal and malignant cells. The read-outs may provide biological insight into cancer pathogenesis, identify novel drug targets, and ultimately serve as a biomarker to guide clinical decision-making.

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127.

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Modulation of proximal signaling in normal and transformed B cells by transmembrane adapter Cbp/PAG.

The transmembrane protein Cbp/PAG (Csk binding protein/phospho-protein associated with glycosphingolipid-enriched microdomains) has a negative regulatory role in T cell activation as an adapter for C-terminal Src kinase, Csk. In T cells, membrane docking of Csk is promoted by binding to FynT-phosphorylated Cbp/PAG (pTyr317) to allow targeting of substrates residing in lipid rafts. Here, we investigate a potential parallel position for Cbp/PAG and the Src kinase Lyn in early B cell receptor signaling. Using normal and transformed B cells, we have compared signal profiles of BCR-triggered responses created by phospho-specific flow cytometry. In human normal B cells, our data show that reduced Cbp/PAG levels leads to enhanced and prolonged activation of proximal signaling mediators, while over-expression of the adapter in normal, EBV-transformed cells results in reduced calcium flux. Taken together, our findings support a negative regulatory function for Cbp/PAG in proximal BCR signaling in these cells.

A tumor microenvironment model of chronic lymphocytic leukemia enables drug sensitivity testing to guide precision medicine.

The microenvironment of chronic lymphocytic leukemia (CLL) cells in lymph nodes, spleen, and bone marrow provides survival, proliferation, and drug resistance signals. Therapies need to be effective in these compartments, and pre-clinical models of CLL that are used to test drug sensitivity must mimic the tumor microenvironment to reflect clinical responses. Ex vivo models have been developed that capture individual or multiple aspects of the CLL microenvironment, but they are not necessarily compatible with high-throughput drug screens. Here, we report on a model that has reasonable associated costs, can be handled in a regularly equipped cell lab, and is compatible with ex vivo functional assays including drug sensitivity screens. The CLL cells are cultured with fibroblasts that express the ligands APRIL, BAFF and CD40L for 24 h. The transient co-culture was shown to support survival of primary CLL cells for at least 13 days, and mimic in vivo drug resistance signals. Ex vivo sensitivity and resistance to the Bcl-2 antagonist venetoclax correlated with in vivo responses. The assay was used to identify treatment vulnerabilities and guide precision medicine for a patient with relapsed CLL. Taken together, the presented CLL microenvironment model enables clinical implementation of functional precision medicine in CLL.

Clinical forecasting of acute myeloid leukemia using ex vivo drug-sensitivity profiling.

Current treatment selection for acute myeloid leukemia (AML) patients depends on risk stratification based on cytogenetic and genomic markers. However, the forecasting accuracy of treatment response remains modest, with most patients receiving intensive chemotherapy. Recently, ex vivo drug screening has gained traction in personalized treatment selection and as a tool for mapping patient groups based on relevant cancer dependencies. Here, we systematically evaluated the use of drug sensitivity profiling for predicting patient survival and clinical response to chemotherapy in a cohort of AML patients. We compared computational methodologies for scoring drug efficacy and characterized tools to counter noise and batch-related confounders pervasive in high-throughput drug testing. We show that ex vivo drug sensitivity profiling is a robust and versatile approach to patient prognostics that comprehensively maps functional signatures of treatment response and disease progression. In conclusion, ex vivo drug profiling can assess risk for individual AML patients and may guide clinical decision-making.

Standardized assays to monitor drug sensitivity in hematologic cancers.

The principle of drug sensitivity testing is to expose cancer cells to a library of different drugs and measure its effects on cell viability. Recent technological advances, continuous approval of targeted therapies, and improved cell culture protocols have enhanced the precision and clinical relevance of such screens. Indeed, drug sensitivity testing has proven diagnostically valuable for patients with advanced hematologic cancers. However, different cell types behave differently in culture and therefore require optimized drug screening protocols to ensure that their ex vivo drug sensitivity accurately reflects in vivo drug responses. For example, primary chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells require unique microenvironmental stimuli to survive in culture, while this is less the case for acute myeloid leukemia (AML) cells. Here, we present our optimized and validated protocols for culturing and drug screening of primary cells from AML, CLL, and MM patients, and a generic protocol for cell line models. We also discuss drug library designs, reproducibility, and quality controls. We envision that these protocols may serve as community guidelines for the use and interpretation of assays to monitor drug sensitivity in hematologic cancers and thus contribute to standardization. The read-outs may provide insight into tumor biology, identify or confirm treatment resistance and sensitivity in real time, and ultimately guide clinical decision-making.

Computational Pipeline for Rational Drug Combination Screening in Patient-Derived Cells.

In many complex diseases, such as cancers, resistance to monotherapies easily occurs, and longer-term treatment responses often require combinatorial therapies as next-line regimens. However, due to a massive number of possible drug combinations to test, there is a need for systematic and rational approaches to finding safe and effective drug combinations for each individual patient. This protocol describes an ecosystem of computational methods to guide high-throughput combinatorial screening that help experimental researchers to identify optimal drug combinations in terms of synergy, efficacy, and/or selectivity for further preclinical and clinical investigation. The methods are demonstrated in the context of combinatorial screening in primary cells of leukemia patients, where the translational aim is to identify drug combinations that show not only high synergy but also maximal cancer-selectivity. The mechanism-agnostic and cost-effective computational methods are widely applicable to various cancer types, which are amenable to drug testing, as the computational methods take as input only the phenotypic measurements of a subset of drug combinations, without requiring target information or genomic profiles of the patient samples.