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.

Finding the Goldilocks Zone of Mechanical Loading: A Comprehensive Review of Mechanical Loading in the Prevention and Treatment of Knee Osteoarthritis.

In this review, we discuss the interaction of mechanical factors influencing knee osteoarthritis (KOA) and post-traumatic osteoarthritis (PTOA) pathogenesis. Emphasizing the importance of mechanotransduction within inflammatory responses, we discuss its capacity for being utilized and harnessed within the context of prevention and rehabilitation of osteoarthritis (OA). Additionally, we introduce a discussion on the Goldilocks zone, which describes the necessity of maintaining a balance of adequate, but not excessive mechanical loading to maintain proper knee joint health. Expanding beyond these, we synthesize findings from current literature that explore the biomechanical loading of various rehabilitation exercises, in hopes of aiding future recommendations for physicians managing KOA and PTOA and athletic training staff strategically planning athlete loads to mitigate the risk of joint injury. The integration of these concepts provides a multifactorial analysis of the contributing factors of KOA and PTOA, in order to spur further research and illuminate the potential of utilizing the body's own physiological responses to mechanical stimuli in the management of OA.

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells.

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how their location can impact their function. In particular, the separation of nuclear and cytoplasmic RNA has become important in the context of cancer cells and the quest to find new targets for drugs. Purchasing kits for nuclear-cytoplasmic RNA extraction can be costly when many of the required materials can be found within a typical lab setting. Using the present method, which can replace more expensive kits or other time-consuming processes, only a homemade lysis buffer, a benchtop centrifuge, and RNA isolation purification columns are needed to isolate nuclear and cytoplasmic RNA. Lysis buffer is used to gently lyse the cell's outer membrane without affecting the integrity of the nuclear envelope, allowing for releasing its intracellular components. Then, the nuclei can be isolated by a simple centrifugation step since they possess a higher density than the lysis solution. Centrifugation is utilized to separate these areas based on their density differences to isolate subcellular elements in the nucleus from those in the cytoplasm. Once the centrifugation has isolated the different components, an RNA clean-up kit is utilized to purify the RNA content, and qPCR is performed to validate the separation quality, quantified by the amount of nuclear and cytoplasmic RNA in the different fractions. Statistically significant levels of separation were achieved, illustrating the protocol's effectiveness. In addition, this system can be adapted for the isolation of different types of RNA (total, small RNA, etc.), which allows for targeted studying of cytoplasm-nucleus interactions, and aids in understanding the differences in the function of RNA that reside in the nucleus and cytoplasm.

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.

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.