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.

Targeted Therapy Development in Acute Myeloid Leukemia.

Therapeutic developments targeting acute myeloid leukemia (AML) have been in the pipeline for five decades and have recently resulted in the approval of multiple targeted therapies. However, there remains an unmet need for molecular treatments that can deliver long-term remissions and cure for this heterogeneous disease. Previously, a wide range of small molecule drugs were developed to target sub-types of AML, mainly in the relapsed and refractory setting; however, drug resistance has derailed the long-term efficacy of these as monotherapies. Recently, the small molecule venetoclax was introduced in combination with azacitidine, which has improved the response rates and the overall survival in older adults with AML compared to those of chemotherapy. However, this regimen is still limited by cytotoxicity and is not curative. Therefore, there is high demand for therapies that target specific abnormalities in AML while sparing normal cells and eliminating leukemia-initiating cells. Despite this, the urgent need to develop these therapies has been hampered by the complexities of this heterogeneous disease, spurring the development of innovative therapies that target different mechanisms of leukemogenesis. This review comprehensively addresses the development of novel targeted therapies and the translational perspective for acute myeloid leukemia, including the development of selective and non-selective drugs.

Protein biomarkers for response to XPO1 inhibition in haematologic malignancies.

XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumour and haematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes, not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using haematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-κB(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision-making in the personalized treatment of cancer patients undergoing treatment with selinexor.

Tracking the evolution of therapy-related myeloid neoplasms using chemotherapy signatures.

Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Preleukemic clones (ie, clonal hematopoiesis [CH]) are detectable years before the development of these aggressive malignancies, although the genomic events leading to transformation and expansion are not well defined. Here, by leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of prechemotherapy samples, we reconstructed the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy was revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan were hypermutated and enriched for complex structural variants (ie, chromothripsis), whereas neoplasms with nonmutagenic chemotherapy exposures were genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to discrete clinical exposure in each patient's life, we estimated that several complex events and genomic drivers were acquired after chemotherapy was administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes melphalan exposure and is selected after reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA damage. Overall, we revealed a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select preexisting CH but also promote the acquisition of recurrent genomic drivers.

Therapy-selected clonal hematopoiesis and its role in myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MN) account for approximately 10-15% of all myeloid neoplasms and are associated with poor prognosis. Genomic characterization of t-MN to date has been limited in comparison to the considerable sequencing efforts performed for de novo myeloid neoplasms. Until recently, targeted deep sequencing (TDS) or whole exome sequencing (WES) have been the primary technologies utilized and thus limited the ability to explore the landscape of structural variants and mutational signatures. In the past decade, population-level studies have identified clonal hematopoiesis as a risk factor for the development of myeloid neoplasms. However, emerging research on clonal hematopoiesis as a risk factor for developing t-MN is evolving, and much is unknown about the progression of CH to t-MN. In this work, we will review the current knowledge of the genomic landscape of t-MN, discuss background knowledge of clonal hematopoiesis gained from studies of de novo myeloid neoplasms, and examine the recent literature studying the role of therapeutic selection of CH and its evolution under the effects of antineoplastic therapy. Finally, we will discuss the potential implications on current clinical practice and the areas of focus needed for future research into therapy-selected clonal hematopoiesis in myeloid neoplasms.

Combination venetoclax and selinexor effective in relapsed refractory multiple myeloma with translocation t(11;14).

Patients with multiple myeloma-bearing translocation t(11;14) have recently been shown to benefit from the apoptosis-inducing drug venetoclax; however, the drug lacks FDA approval in multiple myeloma thus far due to a potential safety signal in the overall patient population. Selinexor is an inhibitor of nuclear export that is FDA-approved for patients with multiple myeloma refractory to multiple lines of therapy. Here, we report that in four patients with multiple myeloma with t(11;14), the concomitant administration of venetoclax and selinexor was safe and associated with disease response. Moreover, the combination was synergistic in t(11;14) multiple myeloma cell lines and caused decreased levels of Cyclin D1 (which is overexpressed due to the CCND1-IGH fusion) when given in combination as compared to single agents. These data suggest that the combination of venetoclax and selinexor is effective and t(11;14) may serve as a therapeutic marker for response and target for future clinical trials.

Urolithin A attenuates severity of chronic pancreatitis associated with continued alcohol intake by inhibiting PI3K/AKT/mTOR signaling.

Heavy alcohol consumption is the dominant risk factor for chronic pancreatitis (CP); however, treatment and prevention strategies for alcoholic chronic pancreatitis (ACP) remains limited. The present study demonstrates that ACP induction in C57BL/6 mice causes significant acinar cell injury, pancreatic stellate cell (PSC) activation, exocrine function insufficiency, and an increased fibroinflammatory response when compared with alcohol or CP alone. Although the withdrawal of alcohol during ACP recovery led to reversion of pancreatic damage, continued alcohol consumption with established ACP perpetuated pancreatic injury. In addition, phosphokinase array and Western blot analysis of ACP-induced mice pancreata revealed activation of the phosphatidylinositol 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and cyclic AMP response element binding protein (CREB) signaling pathways possibly orchestrating the fibroinflammatory program of ACP pathogenesis. Mice treated with urolithin A (Uro A, a gut-derived microbial metabolite) in the setting of ACP with continued alcohol intake (during the recovery period) showed suppression of AKT and P70S6K activation, and acinar damage was significantly reduced with a parallel reduction in pancreas-infiltrating macrophages and proinflammatory cytokine accumulation. These results collectively provide mechanistic insight into the impact of Uro A on attenuation of ACP severity through suppression of PI3K/AKT/mTOR signaling pathways and can be a useful therapeutic approach in patients with ACP with continuous alcohol intake.NEW & NOTEWORTHY Our novel findings presented here demonstrate the utility of Uro A as an effective therapeutic agent in attenuating alcoholic chronic pancreatitis (ACP) severity with alcohol continuation after established disease, through suppression of the PI3K/AKT/mTOR signaling pathway.

Lung Inflammation Predictors in Combined Immune Checkpoint-Inhibitor and Radiation Therapy-Proof-of-Concept Animal Study.

PURPOSE: Combined radiotherapy (RT) and immune checkpoint-inhibitor (ICI) therapy can act synergistically to enhance tumor response beyond what either treatment can achieve alone. Alongside the revolutionary impact of ICIs on cancer therapy, life-threatening potential side effects, such as checkpoint-inhibitor-induced (CIP) pneumonitis, remain underreported and unpredictable. In this preclinical study, we hypothesized that routinely collected data such as imaging, blood counts, and blood cytokine levels can be utilized to build a model that predicts lung inflammation associated with combined RT/ICI therapy. MATERIALS AND METHODS: This proof-of-concept investigational work was performed on Lewis lung carcinoma in a syngeneic murine model. Nineteen mice were used, four as untreated controls and the rest subjected to RT/ICI therapy. Tumors were implanted subcutaneously in both flanks and upon reaching volumes of ~200 mm3 the animals were imaged with both CT and MRI and blood was collected. Quantitative radiomics features were extracted from imaging of both lungs. The animals then received RT to the right flank tumor only with a regimen of three 8 Gy fractions (one fraction per day over 3 days) with PD-1 inhibitor administration delivered intraperitoneally after each daily RT fraction. Tumor volume evolution was followed until tumors reached the maximum size allowed by the Institutional Animal Care and Use Committee (IACUC). The animals were sacrificed, and lung tissues harvested for immunohistochemistry evaluation. Tissue biomarkers of lung inflammation (CD45) were tallied, and binary logistic regression analyses were performed to create models predictive of lung inflammation, incorporating pretreatment CT/MRI radiomics, blood counts, and blood cytokines. RESULTS: The treated animal cohort was dichotomized by the median value of CD45 infiltration in the lungs. Four pretreatment radiomics features (3 CT features and 1 MRI feature) together with pre-treatment neutrophil-to-lymphocyte (NLR) ratio and pre-treatment granulocyte-macrophage colony-stimulating factor (GM-CSF) level correlated with dichotomized CD45 infiltration. Predictive models were created by combining radiomics with NLR and GM-CSF. Receiver operating characteristic (ROC) analyses of two-fold internal cross-validation indicated that the predictive model incorporating MR radiomics had an average area under the curve (AUC) of 0.834, while the model incorporating CT radiomics had an AUC of 0.787. CONCLUSIONS: Model building using quantitative imaging data, blood counts, and blood cytokines resulted in lung inflammation prediction models justifying the study hypothesis. The models yielded very-good-to-excellent AUCs of more than 0.78 on internal cross-validation analyses.

Mechanisms of Resistance to Noncovalent Bruton's Tyrosine Kinase Inhibitors.

BACKGROUND: Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. METHODS: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. RESULTS: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors. CONCLUSIONS: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).

Toward prediction of abscopal effect in radioimmunotherapy: Pre-clinical investigation.

PURPOSE: Immunotherapy (IT) and radiotherapy (RT) can act synergistically, enhancing antitumor response beyond what either treatment can achieve separately. Anecdotal reports suggest that these results are in part due to the induction of an abscopal effect on non-irradiated lesions. Systematic data on incidence of the abscopal effect are scarce, while the existence and the identification of predictive signatures or this phenomenon are lacking. The purpose of this pre-clinical investigational work is to shed more light on the subject by identifying several imaging features and blood counts, which can be utilized to build a predictive binary logistic model. MATERIALS AND METHODS: This proof-of-principle study was performed on Lewis Lung Carcinoma in a syngeneic, subcutaneous murine model. Nineteen mice were used: four as control and the rest were subjected to combined RT plus IT regimen. Tumors were implanted on both flanks and after reaching volume of ~200 mm3 the animals were CT and MRI imaged and blood was collected. Quantitative imaging features (radiomics) were extracted for both flanks. Subsequently, the treated animals received radiation (only to the right flank) in three 8 Gy fractions followed by PD-1 inhibitor administrations. Tumor volumes were followed and animals exhibiting identical of better tumor growth delay on the non-irradiated (left) flank as compared to the irradiated flank were identified as experiencing an abscopal effect. Binary logistic regression analysis was performed to create models for CT and MRI radiomics and blood counts, which are predictive of the abscopal effect. RESULTS: Four of the treated animals experienced an abscopal effect. Three CT and two MRI radiomics features together with the pre-treatment neutrophil-to-lymphocyte (NLR) ratio correlated with the abscopal effect. Predictive models were created by combining the radiomics with NLR. ROC analyses indicated that the CT model had AUC of 0.846, while the MRI model had AUC of 0.946. CONCLUSIONS: The combination of CT and MRI radiomics with blood counts resulted in models with AUCs of 1 on the modeling dataset. Application of the models to the validation dataset exhibited AUCs above 0.84, indicating very good predictive power of the combination between quantitative imaging and blood counts.

Prostate Cancer Targeted X-Ray Fluorescence Imaging via Gold Nanoparticles Functionalized With Prostate-Specific Membrane Antigen (PSMA).

PURPOSE: The gold nanoparticle (GNP) as a promising theranostic probe has been increasingly studied. The tumor-targeting efficiency of GNPs is crucial to increase the therapeutic ratio. In this study, we developed PSMA-targeted GNPs to enhance GNP uptake in prostate cancer and developed an x-ray fluorescence imaging system to noninvasively monitor and assess GNP delivery. METHODS AND MATERIALS: For targeted therapy of prostate cancer, anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated onto PEGylated GNPs through 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC/NHS) chemistry. In vivo imaging was implemented using an in-house-developed dual-modality computed tomography (CT) and x-ray fluorescence CT (XFCT) system on mice bearing subcutaneous LNCaP prostate tumors. After intravenous administration of GNPs (15 mg/mL, 200 µL), the x-ray fluorescence signals from the tumor were collected at various time points (5 minutes to approximately 30 hours) for GNP pharmacokinetics analysis. At 24 hours after administration, x-ray fluorescence projection (XRFproj) and XFCT imaging were conducted to evaluate the prostate tumor uptake of active- and passive-targeting GNPs. Inductively coupled plasma mass spectrometry analysis was adopted as a benchmark to verify the quantification accuracy of XRFproj/XFCT imaging. RESULTS: Fluorescence microscopic imaging confirmed the enhanced (approximately 4 times) targeting efficiency of PSMA-targeted GNPs in vitro. The pharmacokinetics analysis showed enhanced tumor uptake/retention of PSMA-targeted GNPs and revealed that the peak tumor accumulation appeared at approximately 24 hours after intravenous administration. Both XRFproj and XFCT imaging presented their accuracy in quantifying GNPs within tumors noninvasively. Moreover, XFCT imaging verified its unique capabilities to simultaneously determine the heterogeneous spatial distribution and the concentration of GNPs within tumors in vivo. CONCLUSIONS: In conjunction with PSMA-targeted GNPs, XRFproj/XFCT would be a highly sensitive tool for targeted imaging of prostate cancer, benefiting the elucidation of mechanisms of GNP-assisted prostate-cancer therapy.

Radiation Attenuates Prostate Tumor Antiviral Responses to Vesicular Stomatitis Virus Containing IFNß, Resulting in Pronounced Antitumor Systemic Immune Responses.

Vesicular stomatitis virus (VSV) expressing IFNß induces apoptosis in multiple tumor models while maintaining an excellent safety profile. VSV-IFNß is oncoselective due to permissive replication in cells with an altered IFN pathway. The human VSV-IFNß (hIFNß) vector is currently used in clinical trials as a standalone therapy; however, we hypothesized that oncolytic virotherapy might be more effective when used in combination with radiotherapy (RT). We investigated the synergistic effects of RT and VSV-hIFNß in the subcutaneous PC3 and orthotopic LNCaP prostate xenograft models and a syngeneic RM9 prostate tumor model. VSV-IFNß combined with RT amplified tumor killing for PC3 and LNCaP xenografts, and RM9 tumors. This was attributed to the induction of proapoptotic genes leading to increased VSV-IFNß infection and replication, VSV expression, and oncolysis. In the RM9 tumors, combination therapy resulted in a robust antitumor immune response. Treated RM9 tumor-bearing mice demonstrated an increase in CD8+ and CD4+ T-cell numbers, 100% resistance to tumor rechallenge, and reduced resistance to reimplantation challenge with CD8+ knockdown. RT enhanced the activity of VSV-mediated oncolysis via attenuation of the innate antiviral response, resulting in increased VSV replication and the generation of an adaptive immune response earmarked by an increase in CD8+ lymphocyte numbers and antitumor activity. Local tumor irradiation combined with VSV-IFNß affects tumor cell death through direct and systemic activity in conjunction with pronounced antitumor immunity. IMPLICATIONS: Radiotherapy enhances VSV-mediated oncolysis and anti-tumor immunity, indicating that the ombination has promise for very high risk prostate cancer.

Urolithin A, a Novel Natural Compound to Target PI3K/AKT/mTOR Pathway in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and is highly resistant to standard treatment regimens. Targeted therapies against KRAS, a mutation present in an overwhelming majority of PDAC cases, have been largely ineffective. However, inhibition of downstream components in the KRAS signaling cascade provides promising therapeutic targets in the management of PDAC and warrants further exploration. Here, we investigated Urolithin A (Uro A), a novel natural compound derived from pomegranates, which targets numerous kinases downstream of KRAS, in particular the PI3K/AKT/mTOR signaling pathways. We showed that treatment of PDAC cells with Uro A blocked the phosphorylation of AKT and p70S6K in vitro, successfully inhibited the growth of tumor xenografts, and increased overall survival of Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mice compared with vehicle or gemcitabine therapy alone. Histologic evaluation of these Uro A-treated tumor samples confirmed mechanistic actions of Uro A via decreased phosphorylation of AKT and p70S6K, reduced proliferation, and increased cellular apoptosis in both xenograft and PKT mouse models. In addition, Uro A treatment reprogrammed the tumor microenvironment, as evidenced by reduced levels of infiltrating immunosuppressive cell populations such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Overall, this work provides convincing preclinical evidence for the utility of Uro A as a therapeutic agent in PDAC through suppression of the PI3K/AKT/mTOR pathway.

Successful derivation of EGFP-transgenic embryonic stem cell line from a genetically non-permissive FVB/N mouse.

Derivation of embryonic stem (ES)-cell lines from genetically non-permissive mouse strains, such as FVB/N, has been difficult, despite this strain offering advantages for mouse transgenesis for developmental studies. We earlier generated ß-actin promoter-driven enhanced green fluorescent protein (EGFP)-transgenic FVB/N mice, expressing EGFP in all cells. Here, by optimizing culture system and using RESGROTM ES-cell culture medium, we successfully derived EGFP-transgenic ES-cell line, 'GS-2' line, from F1 hybrid blastocysts, from wild-type 129/SvJ female X EGFP-transgenic homozygous FVB/N male. The GS-2 ES-cell line exhibited all defining criteria of a typical ES-cell line, including normal colony morphology and karyotype (40,XY), high replication-expansion efficiency (passages: >100), expression of pluripotent markers (Oct-4, Nanog, Sox-2, SSEA-1 and others) and, embryoid body (EB) development and EB differentiation to ecto-/meso-/endo-dermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively. GS-2 ES-cells formed (i) teratoma containing three germ lineage-derived cell types, (ii) chimeric blastocysts and fetuses, following their aggregation with wild-type 8-cell embryos, (iii) functional cardiac clusters and (iv) predominantly neural cell types when EBs were developed in KOSR-supplemented medium. Taken together, we derived a robust EGFP-transgenic GS-2 ES-cell line, from a non-permissive transgenic (FVB/N) mouse by a single cross to 129/SvJ wild-type mouse. The GS-2 ES-cell line exhibited full differentiation potential, in vitro/in vivo, providing enormous opportunity for stem cell research, including experimental cell transplantation studies.