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.

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.

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.

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.

Mutational analysis and protein profiling predict drug sensitivity in multiple myeloma cell lines.

Introduction: Multiple myeloma (MM) is a heterogeneous disease where cancer-driver mutations and aberrant signaling may lead to disease progression and drug resistance. Drug responses vary greatly, and there is an unmet need for biomarkers that can guide precision cancer medicine in this disease. Methods: To identify potential predictors of drug sensitivity, we applied integrated data from drug sensitivity screening, mutational analysis and functional signaling pathway profiling in 9 cell line models of MM. We studied the sensitivity to 33 targeted drugs and their association with the mutational status of cancer-driver genes and activity level of signaling proteins. Results: We found that sensitivity to mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol-3 kinase (PI3K) inhibitors correlated with mutations in NRAS/KRAS, and PI3K family genes, respectively. Phosphorylation status of MEK1 and protein kinase B (AKT) correlated with sensitivity to MEK and PI3K inhibition, respectively. In addition, we found that enhanced phosphorylation of proteins, including Tank-binding kinase 1 (TBK1), as well as high expression of B cell lymphoma 2 (Bcl-2), correlated with low sensitivity to MEK inhibitors. Discussion: Taken together, this study shows that mutational status and signaling protein profiling might be used in further studies to predict drug sensitivities and identify resistance markers in MM.

Functional precision cancer medicine: drug sensitivity screening enabled by cell culture models.

Functional precision medicine is a new, emerging area that can guide cancer treatment by capturing information from direct perturbations of tumor-derived, living cells, such as by drug sensitivity screening. Precision cancer medicine as currently implemented in clinical practice has been driven by genomics, and current molecular tumor boards rely extensively on genomic characterization to advise on therapeutic interventions. However, genomic biomarkers can only guide treatment decisions for a fraction of the patients. In this review we provide an overview of the current state of functional precision medicine, highlight advances for drug-sensitivity screening enabled by cell culture models, and discuss how artificial intelligence (AI) can be coupled to functional precision medicine to guide patient stratification.

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.

Determining drug dose in the era of targeted therapies: playing it (un)safe?

Targeted therapies against phosphatidylinositol 3-kinase (PI3K), Bruton's tyrosine kinase (BTK), and B-cell lymphoma-2 (BCL-2) are approved for chronic lymphocytic leukemia (CLL). Since approval of the first-in-class drugs, next-generation agents have become available and are continuously under development. While these therapies act on well-characterized molecular targets, this knowledge is only to some extent taken into consideration when determining their dose in phase I trials. For example, BTK occupancy has been assessed in dose-finding studies of various BTK inhibitors, but the minimum doses that result in full BTK occupancy were not determined. Although targeted agents have a different dose-response relationship than cytotoxic agents, which are more effective near the maximum tolerated dose, the traditional 3 + 3 toxicity-driven trial design remains heavily used in the era of targeted therapies. If pharmacodynamic biomarkers were more stringently used to guide dose selection, the recommended phase II dose would likely be lower as compared to the toxicity-driven selection. Reduced drug doses may lower toxicity, which in some cases is severe for these agents, and are supported by retrospective studies demonstrating non-inferior outcomes for patients with clinically indicated dose reductions. Here, we review strategies that were used for dose selection in phase I studies of currently approved and select investigational targeted therapies in CLL, and discuss how our initial clinical experience with targeted therapies have pointed to dose reductions, intermittent dosing, and drug combinations as strategies to overcome treatment intolerance and resistance.

Functional Testing to Characterize and Stratify PI3K Inhibitor Responses in Chronic Lymphocytic Leukemia.

PURPOSE: PI3K inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). Although patients may show an initial response to these therapies, development of treatment intolerance or resistance remain clinical challenges. To overcome these, prediction of individual treatment responses based on actionable biomarkers is needed. Here, we characterized the activity and cellular effects of 10 PI3Ki and investigated whether functional analyses can identify treatment vulnerabilities in PI3Ki-refractory/intolerant CLL and stratify responders to PI3Ki. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cell samples (n = 51 in total) from treatment-naïve and PI3Ki-treated patients with CLL were studied. Cells were profiled against 10 PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay. RESULTS: pan-PI3Kis were most effective at inhibiting PI3K signaling and cell viability, and showed activity in CLL cells from both treatment-naïve and idelalisib-refractory/intolerant patients. CLL cells from idelalisib-refractory/intolerant patients showed overall reduced protein phosphorylation levels. The pan-PI3Ki copanlisib, but not the p110δ inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, but did not significantly affect T-cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Analysis of drug sensitivities to 73 drug combinations and profiling of 31 proteins stratified responders to idelalisib and umbralisib, respectively. CONCLUSIONS: Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and indicate that ex vivo functional profiling may stratify PI3Ki responders.

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.

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.

Ex vivo drug sensitivity screening in multiple myeloma identifies drug combinations that act synergistically.

The management of multiple myeloma (MM) is challenging: An assortment of available drug combinations adds complexity to treatment selection, and treatment resistance frequently develops. Given the heterogeneous nature of MM, personalized testing tools are required to identify drug sensitivities. To identify drug sensitivities in MM cells, we established a drug testing pipeline to examine ex vivo drug responses. MM cells from 44 patients were screened against 30 clinically relevant single agents and 44 double- and triple-drug combinations. We observed variability in responses across samples. The presence of gain(1q21) was associated with low sensitivity to venetoclax, and decreased ex vivo responses to dexamethasone reflected the drug resistance observed in patients. Less heterogeneity and higher efficacy was detected with many combinations compared to the corresponding single agents. We identified new synergistic effects of melflufen plus panobinostat using low concentrations (0.1-10 nm and 8 nm, respectively). In agreement with clinical studies, clinically approved combinations, such as triple combination of selinexor plus bortezomib plus dexamethasone, acted synergistically, and synergies required low drug concentrations (0.1 nm bortezomib, 10 nm selinexor and 4 nm dexamethasone). In summary, our drug screening provided results within a clinically actionable 5-day time frame and identified synergistic drug efficacies in patient-derived MM cells that may aid future therapy choices.

Mcl-1 and Bcl-xL levels predict responsiveness to dual MEK/Bcl-2 inhibition in B-cell malignancies.

Most patients with chronic lymphocytic leukemia (CLL) initially respond to targeted therapies, but eventually relapse and develop resistance. Novel treatment strategies are therefore needed to improve patient outcomes. Here, we performed direct drug testing on primary CLL cells and identified synergy between eight different mitogen-activated protein kinase kinase (MEK) inhibitors and the B-cell lymphoma 2 (Bcl-2) antagonist venetoclax. Drug sensitivity was independent of immunoglobulin heavy-chain gene variable region (IGVH) and tumor protein p53 (TP53) mutational status, and CLL cells from idelalisib-resistant patients remained sensitive to the treatment. This suggests that combined MEK/Bcl-2 inhibition may be an option for high-risk CLL. To test whether sensitivity could be detected in other B-cell malignancies, we performed drug testing on cell line models of CLL (n = 4), multiple myeloma (MM; n = 8), and mantle cell lymphoma (MCL; n = 7). Like CLL, MM cells were sensitive to the MEK inhibitor trametinib, and synergy was observed with venetoclax. In contrast, MCL cells were unresponsive to MEK inhibition. To investigate the underlying mechanisms of the disease-specific drug sensitivities, we performed flow cytometry-based high-throughput profiling of 31 signaling proteins and regulators of apoptosis in the 19 cell lines. We found that high expression of the antiapoptotic proteins myeloid cell leukemia-1 (Mcl-1) or B-cell lymphoma-extra large (Bcl-xL) predicted low sensitivity to trametinib + venetoclax. The low sensitivity could be overcome by combined treatment with an Mcl-1 or Bcl-xL inhibitor. Our findings suggest that MEK/Bcl-2 inhibition has therapeutic potential in leukemia and myeloma, and demonstrate that protein expression levels can serve as predictive biomarkers for treatment sensitivities.

A heterozygous germline CD100 mutation in a family with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease without clear etiology or effective treatment. Genetic factors contribute to PSC pathogenesis, but so far, no causative mutation has been found. We performed whole-exome sequencing in a family with autosomal dominant inheritance of PSC and identified a heterozygous germline missense mutation in SEMA4D, encoding a K849T variant of CD100. The mutation was located in an evolutionarily conserved, unstructured cytosolic region of CD100 affecting downstream signaling. It was found to alter the function of CD100-expressing cells with a bias toward the T cell compartment that caused increased proliferation and impaired interferon-γ (IFN-γ) production after stimulation. Homologous mutation knock-in mice developed similar IFN-γ impairment in T cells and were more prone to develop severe cholangitis when exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Transfer of wild-type T cells to knock-in mice before and during DDC exposure attenuated cholangitis. Taken together, we identified an inherited mutation in the disordered cytosolic region of CD100 resulting in T cell functional defects. Our findings suggest a protective role for T cells in PSC that might be used therapeutically.