Trends in Oncology

Did you know that Chronic Lymphocytic Leukemia (CLL) is a relentless adversary in the realm of hematologic malignancies? Characterized by the excessive accumulation of abnormal B lymphocytes, CLL presents a unique set of challenges for oncologists, hematologists, and cancer researchers. Despite advancements in targeted therapies and immunotherapies, achieving sustained remission remains elusive for many patients.

In this blog, we will explore the critical role preclinical CLL models play in the development of novel therapies. From understanding the disease's biology to facilitating the development of breakthrough treatments, these models are indispensable tools in our fight against CLL.

The Role of Preclinical CLL Models in Cancer Research

Preclinical CLL models are the unsung heroes of CLL research, providing a crucial bridge between laboratory discoveries and clinical applications. These models enable researchers to study the complex biology of CLL in a controlled environment, offering insights that are impossible to glean from human studies alone.

By replicating the disease in animals or in culture with preclinical CLL models, scientists can test the efficacy and safety of potential therapies before they reach clinical trials. This accelerates the drug development process and enhances our understanding of disease mechanisms. In essence, preclinical CLL models are the bedrock upon which modern CLL cancer therapies are built.

Overview of Current Preclinical CLL Models

The landscape of preclinical CLL models is diverse and continually evolving. Each model offers unique advantages and limitations, making choosing the right one for specific research objectives essential.

Cell line-derived Xenograft (CDX) Models
Xenograft models involve transplanting human CLL cells into immunodeficient mice. These preclinical CLL models allow for the study of human-specific disease characteristics and the evaluation of human-targeted therapies. However, the lack of a fully functional immune system in these mice limits the study of immune-based treatments.

Genetically Engineered Mouse Models (GEMMs)
GEMMs are designed to mimic the genetic aberrations found in human CLL. These preclinical CLL models provide a more accurate representation of the disease's progression and response to therapies. They are particularly valuable for studying the genetic and epigenetic factors driving CLL.

Patient-Derived Xenografts (PDXs)
PDXs involve implanting primary CLL cells from patients into mice and serially passaging to obtain stable in vivo models[1]. These preclinical CLL models retain genetic features of the original tumor, making them predictive of clinical outcomes, although the lack of a proficient immune system needs to be considered. 

Using Preclinical CLL Models in Oncology Research 

The latest therapies approved for CLL are a testament to the power of preclinical CLL models in developing revolutionary therapies. Pirtobrutinib (approved by the FDA at the end of 2023[2]) and lisocabtagene maraleucel (approved by the FDA for use in CLL in 2024[3]) were meticulously tested in preclinical CLL models before their clinical debut, ensuring their safety and efficacy in targeting CLL cells.

Pirtobrutinib is a highly selective, noncovalent Bruton tyrosine kinase inhibitor (BTKi). Preclinical testing of pirtobrutinib was conducted in several preclinical CLL models. CLL cell lines were used to assess target engagement, potency, cellular phosphorylation, and other cellular activity of the inhibitor[4, 5, 6]. Further studies in primary CLL cells and xenograft models confirmed pirtobrutinib's ability to kill CLL cells and reduce tumor burden[4, 6].

Lisocabtagene maraleucel is a CD19-targeted CAR-T cell therapy. Preclinical studies of lisocabtagene maraleucel involved in vitro and xenograft models to evaluate its ability to target and eliminate CLL cells[7, 8]. These preclinical CLL models provided crucial data on the therapy's potency, specificity, and potential side effects.

The success of these preclinical trials paved the way for pirtobrutinib and lisocabtagene maraleucel approval and use in clinical settings. These therapies are now transforming the treatment landscape for CLL and other hematologic malignancies.

The Impact and Future of Preclinical CLL Models in Developing Novel Therapies

The impact of preclinical CLL models on the development of new therapies cannot be overstated. They have accelerated the discovery of novel treatments, reduced the risk of adverse effects, and improved patient outcomes. However, the field is far from static.

Advancements in Model Precision
The future of preclinical CLL models lies in their ability to reproduce clinical characteristics and support personalized medicine. The use of primary patient-derived CLL models by direct injection of patients’ cells in mice without additional passages in the animals allows better preservation of tumor heterogeneity and patient population diversity[9], Patient-derived CLL models can be used in a preclinical trial format as well as for the testing of therapies on individual patient tumors, enabling tailored treatment strategies. 

The development of such models in humanized mice, which possess a reconstituted human immune system, will further improve the robustness of these models as patients’ surrogates by enabling a deeper understanding of how therapies interact with the human immune system, leading to more effective treatments.

Integration of Computational Models
Integrating computational models with preclinical studies is another promising avenue. By simulating disease progression and treatment responses in silico, researchers can optimize experimental designs and predict outcomes more accurately. This synergy between computational and experimental approaches is poised to accelerate the development of next-generation CLL therapies.

The Importance of Continued Research and Innovation

Preclinical CLL models are the linchpin of CLL therapy development, providing invaluable insights and accelerating the transition from bench to bedside. As we continue to refine these models and integrate new technologies, the future of CLL treatment looks increasingly promising.

Champions Oncology's bank of preclinical CLL models includes primary models derived from pretreated and naive patients. With deep multi-omic and multimodal characterization and comprehensive clinical annotations, we strive to make our preclinical CLL models the best tool to accelerate your drug pipeline through reliable data and extensive expertise in the hematologic malignancies field. Contact us to speak with one of our experts. 

Mantle cell lymphoma (MCL) is a rare and aggressive non-Hodgkin’s lymphoma (NHL) that originates in B cells and occurs in secondary follicles of the lymph nodes.¹ MCL accounts for roughly 6% of all lymphomas diagnosed annually, and despite its relatively low incidence, the aggressive course of MCL means it can quickly become a life-threatening disease. Understanding the origin and progression of MCL is crucial to identify new targets for drug development studies.

Understanding Mantle Cell Lymphoma Pathology

Over the last decade, our understanding of MCL pathogenesis has evolved from a single definition to a consensus that MCL development and progression is related to a range of molecular events. Early on MCL was defined as a pathognomonic chromosomal translocation t(11;14)(q13;q32) causing a mutation in the CCND1 gene, resulting in the over-expression of Cyclin D1.^{1, 2}

Under normal conditions, Cyclin D1 is heavily regulated and modulates cell cycle transition from G1 phase to S phase.¹ However, overexpression of Cyclin D1 activates cyclin-dependent kinases (CDK) 4 and 6 which later deactivate the retinoblastoma protein (Rb), a cell cycle inhibitor. This series of events accelerates cell cycle progression from G1 to S phase in many cell types, including B-cells.³ The progression of this process induces uncontrolled B-cell proliferation causing an enlargement of lymph nodes and immune system dysfunction that eventually spreads to critical organs.

Fewer Mutations Raise More Questions

Interestingly, Cyclin D1 mutations are not found in all MCL cells, suggesting other molecular actors, such as transcription factors, are involved. In 90% of MCL patients, transcription factor SOX11 is over-expressed in MCL cells with and without mutated CCND1. While SOX11’s role in MCL is not well understood, studies suggest that PAX-5, a transcription factor that regulates B-cell development and differentiation, is activated by SOX11.⁴ The overexpression of SOX11 that is commonly seen in MCL patients can therefore lead to reduced B-cell differentiation and increased B-cell antigen signaling.^{3, 5}

Identifying Treatment Targets in Mantle Cell Lymphoma

B-cell proliferation and differentiation rely on B-cell receptor (BCR) signaling and are activated in response to antigen binding.⁶ Using the framework for CLL pathogenesis, preliminary MCL-BCR research has found that BCR signaling is highly active in MCL patients and intentional BCR activation resulted in an increase of BCR signaling in MCL cells.⁷ Additionally, murine studies have found SOX11-overexpressing B-cells have high levels of BTK, a key enzyme involved with BCR signaling, resulting in proliferation which suggests SOX11’s deeper role in MCL.⁷

Initial MCL treatment includes R-CHOP therapy, however, many MCL patients are refractory or become resistant creating a need for additional therapies. As with CLL, BCR signaling plays a critical role in MCL progression shifting treatment protocols towards BTK inhibitors (BTKi) like ibrutinib, approved in 2013.^{1, 3} Ibrutinib is metabolized in the liver via CYP3A and CYPRD6 and irreversibly binds to cysteine residue 481 found on the active site of BTK.

While this targeting strategy effectively blocks BTK signaling limiting MCL cell development, ibrutinib has significant off-target consequences, namely IL-2 inducible kinase (ITK), which sparked the development of zanubrutinib.⁸ Zanubrutinib was approved for use in MCL patients and though it has a similar mechanism of action to ibrutinib, its reduced affinity for ITK, a major T-cell and natural killer (NK) cell regulator,⁹ makes it a highly selective and potent BTKi with improved overall response rate relative to ibrutinib.⁸

Resistant Mantle Cell Lymphoma and Future Therapeutic Strategies

Unfortunately, 32% of MCL patients become resistant to BTKi due to BTK mutations.^{10, 11} As a result, combination therapies such as ibrutinib combined with cirmtuzumab (a novel anti-ROR1 monoclonal antibody) or with obinutuzumab and venetoclax have been used to treat resistant MCL patients. ¹¹ These therapeutic combinations have shown excellent remission results paving the way for other combination therapies, such as acalabrutinib, rituximab, and bendamustine. ^{11, 12} Notably, a phase 1 study investigating acalabrutinib, rituximab, and bendamustine achieved an 85% overall response rate which has led to a larger, ongoing Phase 2 study (NCT04115631). Additionally, novel MCL-1 inhibitors are under development in preclinical studies.¹³

Recently, the FDA has approved the first CAR T cell therapy for relapsed/refractory MCL, brexucabtagene autoleucel (Tecartus).¹⁴ Tecartus has shown durable response at the 3-year follow-up even in high-risk patients (NCT02601313), with sustained survival and a 67% complete response rate.¹⁵

Understanding the pathophysiology of naïve and BTKi resistant MCL is crucial to developing curative MCL therapies. While standard of care treatments like ibrutinib and zanubrutinib have had some success, additional studies investigating strategic combination therapies are underway providing hope for patients battling this disease.

Champions supports your in vivo preclinical studies with low passaged MCL PDX models available for subcutaneous modeling and fully characterized with NGS data in 4 models CTG-3771, CTG-3772, CTG-3776 (shown to be rituximab and ibrutinib resistant) and CTG-3808. Preclinical hematological scientists need to evaluate their pipeline of therapeutic candidates in robust hematological screening platforms; Champions' VitroScreen platform can advance potential next-generation therapies into the clinic, generating additional options for MCL patients.   

Acute myeloid leukemia (AML) is the most common leukemia among adults and has been challenging to treat with modern therapies.^{1, 2} This disease is highly heterogeneous and characterized by the rapid proliferation of undifferentiated myeloid cells that accumulate within the bone marrow. Several mutations and epigenetic abnormalities characteristic of AML have been targeted through chemotherapies or molecular inhibitors. Fortunately, the relentless pursuit of innovative, effective AML therapies has led to a deeper understanding of AML pathogenesis, such as the role of isocitrate dehydrogenase (IDH) mutations.³ IDH mutations have unique properties that, if properly targeted, may reshape AML patient outcomes and survival rates.

Unstable Gene Expression

The IDH family consists of three isoenzymes (IDH1, IDH2, and IDH3) and has an important role in the biosynthesis of metabolites involved in the tricarboxylic acid (TCA) cycle. Notably, IDH1 functions as a catalyst for reversible conversion of isocitrate to α-ketoglutarate in the cytosol as well as peroxisomes, yielding 1 NADPH. Downstream, α-ketoglutarate is reduced to D-2-hydroxyglutarate (D-2-HG) as part of the TCA cycle.³ Mutations in IDH1 are present in ~20% of AML patients and result from a single amino acid substitution at Arg132.⁴ This mutation induces an end-product shift that reduces α-ketoglutarate to R-2-HG instead. Unlike D-2-HG, R-2-HG competitively inhibits α-ketoglutarate-dependent enzymes, such as the TET family, and upon accumulation leads to impaired cellular differentiation and deregulation of DNA methylation. ^{3, 5} This alteration of DNA methylation and ultimately gene expression activates oncogenes and deactivates tumor-suppressor genes.⁶ The destabilizing features of IDH1 mutants have thus made this isoenzyme an interesting target for AML therapies.

IDH Focused Therapies

Since the identification of IDH mutations in 2008, the Food and Drug Administration (FDA) has approved two therapeutics for AML patients with IDH mutations, ivosidenib and enasidenib. Ivosidenib is a reversible, selective inhibitor that binds to the active site of mutated IDH1 to prevent the production of R-2-HG.^{7, 8, 9} Reduction in R-2-HG reportedly increases D-2-HG concentrations by 100x and restores DNA methylation and cellular differentiation in AML patients.^{10, 11} Though successful, resistance to ivosidenib and IDH inhibition has emerged, underlining the need for combination therapies that prevent IDH resistance.¹²

Triplet Therapy

There are no triplet regimens currently approved for use in AML, however, clinical trial results have demonstrated successful remission in relapsed and naïve AML patients.^{13, 14} For instance, an ongoing phase Ib/II study examining ivosidenib and venetoclax with or without azacytidine has shown successful remission in patients with AML IDH1 mutations.¹³ The durable response to this therapy is especially promising, as the safety profile of this triplet therapy is similar to doublet therapies such as azacytidine + venetoclax and azacytidine + ivosidenib.^{13, 15} The median event-free-survival (EFS) for patients treated with this new triplet therapy was 36 months¹³, while previous azacytidine + ivosidenib combination treatments have achieved median EFS or 24 months.¹⁶

The Therapeutic Pipeline

Prolonged EFS in AML patients receiving ivosidenib in combination with AML standards of care, such as venetoclax and azacytidine, has recently gained the attention of the European Commission (EC). In 2023, the EC announced the approval of ivosidenib in combination with azacitidine for AML patients with an IDH1 R132 mutation.¹⁸ Further, clinical studies evaluating the side effects and appropriate dosages of ivosidenib and venetoclax with or without azacytidine are underway. As more clinical data is released, access to more robust therapeutic treatments will become available to improve AML patient outcomes.

Hematologic malignancies include a wide array of lymphomas and leukemias that affect different immune cell subsets. Acute myelogenous leukemia (AML) is one of the most commonly occurring leukemias in adults and children. AML is a highly heterogeneous disease that can be caused by spontaneous gene mutations or chromosomal translocations, which result in the proliferation of dysfunctional myeloid cells. Cytogenetic and morphologic analyses have been the gold standard methods used in AML diagnosis. Still, flow cytometry-based protocols are becoming more widely used and validated as complementary diagnostic methods that can be coupled with these analyses to better guide treatment plans. Flow cytometry has also become an essential tool to understand AML progression and develop and evaluate novel therapeutics.

Consider these aspects of flow cytometry-based analysis of AML for exploratory or preclinical research:

Phenotype matters: Immunologists know a lot about what a normally developing myeloid cell should look like in terms of its immunophenotype. Changes in expression of different lineage markers correlate strongly with AML progression and are characterized by the presence of blasts (leukemic cells) in the bone marrow. Flow cytometry-based immunophenotyping offers researchers a rapid and sensitive method for detecting blasts at the onset of disease as well as monitoring changes in this population throughout an experimental therapy.

Rare cells from precious samples: A major consideration of following AML progression is the ability to detect relatively rare cells in bone marrow aspirates. This type of sample may be relatively small and is typically used fresh for morphologic evaluation, but even a small volume of remaining aspirate can be used for flow cytometry-based methods that are sensitive and robust enough to detect blasts.

Paired samples: Bone marrow cells can be analyzed along with peripheral blood using advanced flow cytometry methods that monitor the persistence of blasts and other leukemic subsets over time in these compartments. This type of analysis offers critical insight into the development of new therapeutics whether they are being evaluated in humanized mouse models or clinical trial participants.

Flow cytometry continues to advance immuno-oncology research, especially for diseases involving the detection of rare cell populations. Consider working with preclinical and clinical flow cytometry experts to develop protocols for future immuno-oncology studies.

Multiple myeloma (MM) is the second most common hematological cancer^[1] and results from a series of mutations that lead to malignant plasma cells in the bone marrow.^[2] The survival of MM ranges from 5 to 7 years on average, although patients with high-risk MM, defined as inactive TP53 or 1q21 activation, have reduced survival of 2 years.^{[3, 4]} Due to the complexity of this disease, MM remains incurable, however, the emergence of CAR-T (chimeric antigen receptor T-cell) therapy has provided hope for MM patients.

Pathogenesis

Under normal conditions, plasma cells secrete polyclonal protein or immunoglobulins comprised of one heavy chain and one light chain.^[5] MM is derived from a series of mutations in plasma cells and clonal plasmacytes that instead produce monoclonal proteins (M-proteins) within the bone marrow (BM).^[6] This disease typically progresses from monoclonal gammopathy of undetermined significance (MGUS) that later develops into smoldering multiple myeloma (SMM) – both of which are asymptomatic plasma cell disorders with no end-organ damage.^[7] MGUS is defined as a lower percentage of M-protein (<3g/dl) while SMM is defined as M-protein concentration exceeding 3g/dl.^[8] It is thought that initial mutation events seen in MGUS, and ultimately in MM, that lead to M-protein secretion occur during somatic hypermutation or class-switch recombination events, namely translocations involving IgH locus and IgL locus.^[7] The results of these mutations form an abundance of dysfunctional plasma cells within the bone marrow leading to reduced immune system responses and organ damage.

Therapeutics

Standard of care treatment for newly diagnosed MM includes a cocktail of high-dose chemotherapy agents: bortezomib, lenalidomide, and dexamethasone.^[9] These chemotherapeutic agents are effective at damaging tumor cell DNA as well as tumor microenvironment. Following this aggressive combination therapy, patients undergo allogeneic stem cell transplantation (ASCT) to restore blood cell production.^{[10, 11]} Despite the success of these treatments, many MM patients experience graft-versus-host disease (GVHD) and/or relapse,^[12] making it essential for new and innovative treatments to emerge.

One treatment that has shown promise is chimeric antigen receptor T cell (CAR-T) therapy. CAR-T therapy is a type of immunotherapy that genetically modifies the patient's own T cells to target and kill cancer cells. ^{[12, 13]} In MM patients specifically, B cell maturation antigen (BCMA) targeted CAR-T therapy has been highly successful in newly diagnosed and relapsed MM patients. ^[13] Furthermore, clinical studies have found that CAR-T therapy has a 76% remission rate and conclude better 5-year patient outcomes.^[14] Today there are two FDA-approved CART-T therapies for MM, Carvykti and Abecma, with others in development.^[15] Despite successes, there are notable challenges with CAR-T therapy including cytokine release syndrome (CRS) and neurotoxicity, which may be managed with appropriate and timely interventions. ^[13] CAR-T therapy is also costly and requires specialized manufacturing facilities, limiting its availability to patients.

Importantly, new treatments outside of CAR-T therapy are also in development with novel targets, namely Selinexor. Selinexor targets Chromosome Region Maintenance 1 (XP01), a transporter of nuclear proteins with a key role in cell cycle regulation and proliferation.^[16] XP01 has been implicated in several solid tumors, such as lung cancer, and is overly expressed in MM. Overexpression of XP01 allows tumor suppressor proteins, such as Rb, p53, and p21, to be exported into the cytoplasm rendering them inactive and therefore increasing anti-apoptotic cell signaling.^{[16, 17]} Selinexor covalently binds to the “cargo-binding” groove of XP01 which prevents the displacement of tumor suppressor proteins mentioned above.^{[17, 18]} As a result of its pre-clinical and clinical trial success, the FDA approved Selinexor as a combination therapy with dexamethasone for patients with more than one prior therapy and at least 4 prior dexamethasone therapies.^{[19, 20]}

While MM continues to be a challenging cancer to treat, therapies such as CAR-T therapy and Selinexor provide MM patients with new hope for lasting remission. New clinical trials, such as NCT05177536 to test iberdomide maintenance therapy, are underway to explore the efficacy of new therapies and interventions to improve MM outcomes.

Myelodysplastic syndromes (MDS) are a group of hematological malignancies that manifest in hematopoietic stem cells and are caused by ineffective hematopoiesis.^[1] Currently, MDS is defined as unexplained cytopenia combined with abnormalities in cell maturation leading to dysplastic features in >20% of myeloid cells.^[2] MDS is one of the most frequently diagnosed malignancies in the United States and progresses to acute myeloid leukemia (AML) in 30% of patients.^[2] 

While the initiation of primary MDS is not well understood, research suggests that somatic DNA injury, defective DNA repair, impaired immunological responses, and dysfunctional cell signaling play an important role in early stage MDS development.^[3] Like other malignancies, MDS is positively selected for through gene mutations^[4] with the average MDS patient carrying nine somatic mutations, such as TET2, TP53, and RUNX1.^{[4, 5]} These mutations may not directly drive MDS, however, evidence indicates that mutations to genes involved with DNA methylation and RNA splicing greatly contribute to dysregulation of genes critical to hematopoiesis, such as GATA1, KLF1, and HOXA9.^[6] 

MDS is classically characterized as hematopoietic stem cells (HSC) with clonal advantages due to somatic mutations or cellular dysfunction, discussed above. Studies have also identified the critical role of bone marrow microenvironments (BME) in MDS progression, particularly cytokine alterations and activities. Across several studies, high serum levels of TNF-α, TGF-β, IL-6, and IL-8^[7] are present in MDS patient bone marrow (BM).^[8] These proinflammatory molecules are typically associated with higher apoptotic rates, and in MDS patients, molecules such as TNF- α, correlate to poor MDS treatment performance.^[7] 

Furthermore, studies have also identified the critical role of malignant clones in MDS pathogenesis. Mesenchymal stromal cells (MSCs) are a key component of the bone marrow that regulate hematopoiesis and have immunomodulatory properties. In contrast, dysplastic MSCs in the bone marrow can create a microenvironment that supports clonal expansion of malignant cells. Dysplastic MSCs from MDS or AML patients have phenotypic abnormalities, including aberration in secreted proteins and cell surface protein expression, as well as increased senescence and decreased survival.^[9,10] Disrupted methylation profiles are observed in both MDS malignant clones and MDS-MSCs. These methylation changes impact multiple signaling pathways which create a chronically inflamed BME that is harmful to normal HSCs and supports the expansion of MDS clones.^[11] 

While less understood, de novo MDS has been linked to chemotherapy, radiotherapy, and environmental carcinogens like benzene.^[12] Therapy-related MDS (tMDS) is prevalent in 10-20% of patients 20 years after chemotherapy and/or radiation, and the World Health Organization (WHO) recognizes alkylating agents, like topoisomerase II inhibitors, as initiators of tMDS.^[12] Due to the high incidence of MDS in elderly populations^[13] with co-morbidities, treatment by bone marrow transplantation is often contraindicated and few therapeutic options exist.  

Patients with MDS are typically divided into two different categories: low-risk MDS and high-risk MDS. The division of MDS into these two groups is useful for researchers and clinicians developing therapies for this highly heterogeneic disease.^[14] Low-risk MDS therapies target symptoms of cytopenia through the use of erythropoiesis stimulating agents (ESA), such as epoetin alfa and darbepoetin alfa.^[15] The Food and Drug Administration (FDA) has also approved two hypomethylating agents (HMAs), 5-azacitidine and decitabine, for use in patients with low-risk and high-risk MDS. While both standards of care noncompetitively inhibit DNA methyltransferase (DNMT1) and promote hypomethylation of DNA to block DNA synthesis,^[16] 5-azacitidine integrates with RNA and interferes with ribosomal assembly to also limit tumor protein synthesis.^{[16, 17]}  

In 2023, the FDA and European Medicines Agency (EMA) also approved luspatercept, a recombinant fusion protein that enhances late-stage erythroblast differentiation, for low-risk MDS. Luspatercept indirectly moderates ineffective erythropoiesis by interacting with broad spectrum inhibitory signals, namely transforming growth factor-β (TGF- β) superfamily signaling.^{[18, 19]} Downstream, signaling molecules, SMAD2 and SMAD3, negatively regulate TGF- β. Luspatercept works by binding to activin receptor type IIB on TGF- β which disrupts SMAD2 and SMAD3 signaling therefore improving erythropoiesis and boosting RBC production in MDS patients.^{[20, 21]} 

As our understanding of MDS pathophysiology improves, more effective treatment options will become available. Researchers are actively evaluating the toxicity of allogeneic hematopoietic stem cell transplantation, a therapy with curative potential.^[19] Given the nature of such treatment which involves conditioning chemotherapy, the risk of aplasia and other graft-versus-host disease need to be adequately assessed. Additional studies, such as a newly announced Montefiore Einstein Comprehensive Cancer Center clinical trial,^[22] are investigating new therapies to develop novel therapeutic strategies. 

Chronic lymphocytic leukemia (CLL) is one of the most common forms of adult leukemia, and its chronic nature has made it a challenging blood cancer to completely cure. CLL affects B cells and is typically classified into two categories: little or no somatic hypermutation in the immunoglobulin heavy chain variable region (IGHV), called unmutated CLL, or high mutation levels in the IGHV gene, called mutated CLL.^[1] Unmutated CLL is more aggressive than mutated CLL, and the presence of these abnormal IGHV sequences, a part of B cell receptors (BCR), leads to abnormal BCR signaling and the uncontrolled proliferation of leukemic cells. 

Bruton’s tyrosine kinase (BTK) is an essential enzyme downstream from the BCR and is responsible for propagating the signaling cascade initiated by BCR engagement with pathogen-associated antigens.^[2] Notably, BTK has been shown to be constitutively active in CLL and drives both leukemic cell proliferation and lymph node homing.^[3] As antigen binds to the BCR, multiple protein tyrosine kinases are activated via interactions with the cytoplasmic domain of the BCR, including Lyn and Syk, as well as translocation of BTK to the plasma membrane through interactions with phosphatidylinositol-3,4,5 (PIP3).^[4] Lyn and Syk phosphorylate BTK to activate multiple non-receptor protein tyrosine kinase signaling pathways, including NF-κB, MAPK, and phospholipase C gamma (PLCγ). Under normal conditions, this pathway leads to controlled B cell proliferation and differentiation, however, this same pathway leads to uncontrolled B cell proliferation in malignancies, such as CLL.

The role of BTK in CLL has drawn researchers to develop BTK inhibitors, such as ibrutinib - an FDA (Food and Drug Administration)^[5] and EMA (European Medicines Agency)^[6] - approved CLL treatment. Ibrutinib forms a covalent bond with a cysteine residue (C481) at BTK’s active site, and thus inhibits kinase activity, including autoactivation.^[3] This kinase inhibition shuts down BCR signaling, reduces B cell proliferation, and promotes apoptosis of leukemic cells. Ibrutinib is effective as a standalone treatment for mutated and unmutated CLL and when combined with rituximab is an effective therapy for relapsed CLL.^[1] In general, ibrutinib is well tolerated and shows continued efficacy during extended treatment periods.^[7] 

While ibrutinib has been a clinical success, there are a subset of CLL patients who develop resistance to this therapeutic. This resistance has been mapped to a C481S mutation that prevents ibrutinib from covalently binding to BTK resulting in continuous BCR signaling within leukemic cells.^[3] Next-generation, reversible BTK inhibitors, such as ARQ 531, are currently being developed for relapsed/refractory CLL. ARQ-531 is a non-selective BTK inhibitor that suppresses signaling in cells with C481S BTK and PLCγ mutations. ARQ-531 also has an additional inhibitory activity against ERK signaling.^[8] Furthermore, the FDA has recently approved LOXO-305, a non-covalent, reversible BTK inhibitor. LOXO-305 inhibits signaling in cells with wild-type or C481S-mutated BTK.^{[9, 10]} In contrast, acalabrutinib, a second-generation irreversible BTK inhibitor has also been approved by the FDA. Acalabrutinib has a shorter half-life, allowing for variable dosages, and has increased C481 specificity that enhances its BTK inhibitory effects. The biochemical properties of acalabrutinib make it a suitable option for patients with treatment naïve CLL.^[11] 

In January 2023, the FDA and EMA approved an additional second-generation irreversible BTK inhibitor, zanubrutinib, for CLL.^[12] Zanubrutinib’s design was guided by a structure-activity strategy to generate sustained BTK occupancy. Zanubrutinib exhibits reduced ITK and EGFR inhibition and has 4x longer half-life than acalabrutinib.^[13] As such, it persists at high concentrations within the body making it available to re-inhibit newly synthesized BTK proteins, a unique difference from ibrutinib and acalabrutinib.^[14] 

In a phase I/II study, all CLL patients treated with zanubrutinib had complete and sustained BTK occupancy in peripheral blood mononuclear cells and lymph nodes.^[15] Zanubrutinib’s performance supports the hypothesis that increased BTK selectivity maximizes BTK inhibition and drug efficacy. Additionally, zanubrutinib’s enhanced BTK specificity reduces the incidence of off-target toxicities which may reduce side effects commonly associated with ibrutinib, such as cardiac events, subdural hematomas, and gastrointestinal bleeding.^{[10, 16, 17]} This is particularly relevant to CLL as this class of BTK inhibitors are used by CLL patients indefinitely. 

BTK inhibitors have become an invaluable CLL treatment and will continue to improve and provide therapeutic benefits. Additional studies such as NCT03734016 are underway to evaluate the effects of zanubrutinib and BTK inhibitors with other targeted therapies, which may improve the efficacy and durability of these treatments.  

Recent advances in immunotherapy have led to the development of targeted therapies for treating lymphoma and leukemia. One such therapy is CD19-targeted therapy, which is widely used as a frontline treatment for B cell lymphoma and leukemia. However, despite many patients initially respond well to this therapy relapse remains the major obstacle to be addressed. In this blog post, we will explore the various mechanisms underlying relapse to CD19-targeted therapies in patients with B cell lymphoma and leukemia.

Antigen-positive Relapse:

Early relapse is usually associated with short CAR-T cell persistence. This can be due to poor T cell quality, but, more importantly, it is determined by the costimulatory domain in the CAR. In fact, domains such as 4-1BB have been proven to significantly prolong the persistence of CAR-T cells_[1]. In addition, the gene editing technology used to engineer the T cells can make a difference in CAR-T cell persistence. Combination therapies with checkpoint inhibitors have also been shown to prolong response duration and remission_[1,2].

Immune Evasion Mechanisms:

One of the primary mechanisms of acquired resistance to CD19-targeted therapies is immune evasion. Cancer cells can develop various mechanisms to evade immune surveillance and avoid being targeted by the therapy. For example, cancer cells may downregulate the expression of CD19 on their surface, thereby reducing the effectiveness of the therapy. Alternatively, they may upregulate other immune checkpoint molecules or express inhibitory proteins that prevent immune cells from recognizing and eliminating them_[3].

Antigen Loss Mechanisms:

Another mechanism of acquired resistance is antigen loss. CD19-targeted therapies are designed to bind to and eliminate cancer cells that express the CD19 antigen. However, cancer cells can develop mutations that lead to loss of CD19 expression, rendering the therapy ineffective. This mechanism of resistance has been observed in both lymphoma and leukemia patients, and it is a significant challenge in the development of novel therapies_[4].

Development of Alternative Pathways:

Cancer cells that survive CD19-targeted therapies can develop alternative pathways for survival and growth. These pathways can bypass the effects of the therapy and allow cancer cells to continue to proliferate. Examples of such mechanisms include the activation of alternative signaling pathways and the upregulation of survival factors. Understanding these adaptive mechanisms is crucial in devising new therapeutic strategies to overcome acquired resistance_[1,3].

Tumor Microenvironment:

The tumor microenvironment (TME) plays a critical role in acquired resistance to CD19-targeted therapies. The TME is a complex milieu of immune and stromal cells that interact with cancer cells to promote tumor growth and survival. The TME can also influence the response of cancer cells to therapy. For example, the presence of immunosuppressive cells in the TME can reduce the effectiveness of CD19-targeted therapies_[1,5].

Genetic Factors:

Genetic factors also play a role in acquired resistance to CD19-targeted therapies. For example, mutations in genes involved in DNA repair pathways can increase genomic instability and promote drug resistance. Identifying genetic factors that contribute to resistance can help to personalize therapy and improve outcomes_[6].

Disease relapse and development of acquired resistance to CD19-targeted therapies in patients with B cell lymphoma and leukemia pose a significant clinical challenge. To overcome this challenge, we need to find ways to optimize CAR-T cell design to produce more effective therapies and understand the various mechanisms of resistance to be able to develop new therapeutic strategies that target these mechanisms. Research efforts are underway to identify novel therapeutic targets and personalized treatment approaches that can overcome acquired resistance and improve outcomes for patients_[7].

Continuous improvements in our understanding of acquired resistance and relapse to CD19-targeted therapies will pave the way for the development of more effective and durable treatments for these diseases. To overcome this challenge, access to relevant preclinical models is key to develop alternative or synergistic therapeutic approaches. Champions Oncology offers a large cohort of B-cell lymphoma and leukemia models, including primary samples from patients who progressed after CD19-targeted therapies, to accelerate the development of novel therapeutic approaches to improve B-cell lymphoma and leukemia patients' lives.

1. Shah NN, et al. Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol. 2019 Jun;16(6):372-385. doi: 10.1038/s41571-019-0184-6. PMID: 30837712; PMCID: PMC8214555.
2. Sterner RC, et al. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021 Apr 6;11(4):69. doi: 10.1038/s41408-021-00459-7. PMID: 33824268; PMCID: PMC8024391.
3. Plaks V, et al. CD19 target evasion as a mechanism of relapse in large B-cell lymphoma treated with axicabtagene ciloleucel. Blood. 2021 Sep 23;138(12):1081-1085. doi: 10.1182/blood.2021010930. PMID: 34041526; PMCID: PMC8462361.
4. Sworder BJ, et al. Determinants of resistance to engineered T cell therapies targeting CD19 in large B cell lymphomas. Cancer Cell. 2023 Jan 9;41(1):210-225.e5. doi: 10.1016/j.ccell.2022.12.005. Epub 2022 Dec 29. PMID: 36584673; PMCID: PMC10010070.
5. Frey NV, et al. Optimizing Chimeric Antigen Receptor T-Cell Therapy for Adults With Acute Lymphoblastic Leukemia. J Clin Oncol. 2020 Feb 10;38(5):415-422. doi: 10.1200/JCO.19.01892. Epub 2019 Dec 9. PMID: 31815579; PMCID: PMC8312030.
6. Chen GM, et al. Characterization of Leukemic Resistance to CD19-Targeted CAR T-cell Therapy through Deep Genomic Sequencing. Cancer Immunol Res. 2023 Jan 3;11(1):13-19. doi: 10.1158/2326-6066.CIR-22-0095. PMID: 36255409; PMCID: PMC9808313.
7. Atilla PA, et al. Resistance against anti-CD19 and anti-BCMA CAR T cells: Recent advances and coping strategies. Transl Oncol. 2022 Aug;22:101459. doi: 10.1016/j.tranon.2022.101459. Epub 2022 May 23. PMID: 35617812; PMCID: PMC9136177.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy and the most common leukemia among the adult population. Despite the development of novel targeted therapies, resistance to treatments and disease relapse remain unsolved. New interest in the role of circular RNA in AML biology has opened the way for the development of new approaches in the management of AML.

Somatic mutations in the additional sex comb-like 1 (ASXL1) gene have been identified in multiple hematologic malignancies, including acute myeloid leukemia (AML), and are associated with poor prognoses¹. ASXL1 encodes a nuclear protein that regulates epigenetic remodeling and transcription through interactions with polycomb complex proteins and transcriptional activators and repressors. Several ASXL1 mutations have been associated with loss of protein expression that leads to myeloid transformation². In contrast, gain-of-function mutations in ASXL1 result in expression of a truncated ASXL1 protein that can bind to BRCA-1 associated protein 1 (BAP1) and cause leukemogenesis^3,4. Targeted reduction of BAP1 activity is sufficient to prevent this malignancy process⁵.

A recent study has shown that the ASXL1 gene locus undergoes alternative splicing to produce circular RNAs (circRNAs) in addition to linear protein-coding mRNAs⁶. CircRNAs are non-coding RNAs that normally function as regulators of gene expression and translation by acting as sponges for microRNAs or forming complexes with RNA-binding proteins. Here they identified two isoforms of circular ASXL1 (circASXL1), and they showed that circASXL1-1 can bind to BAP-1 and regulate BAP1-mediated deubiquitinase activity, which targets H2AK119 ubiquitination and regulates myeloid differentiation of hematopoietic stem cells.  These findings suggest that circRNAs may be developed as novel therapeutics for treating hematologic malignancies like AML.
       

1. Pratcorona M, Abbas S, Sanders MA, et al. Acquired mutations in ASXL1 in acute myeloid leukemia: prevalence and prognostic value. Haematologica. 2012;97(3):388-392.
2. Abdel-Wahab O, Adli M, LaFave LM, et al. ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression. Cancer Cell. 2012;22(2):180-193.
3. Balasubramani A, Larjo A, Bassein JA, et al. Cancer-associated ASXL1 mutations may act as gain-of-function mutations of the ASXL1-BAP1 complex. Nat Commun. 2015;6:7307.
4. Asada S, Goyama S, Inoue D, et al. Mutant ASXL1 cooperates with BAP1 to promote myeloid leukaemogenesis. Nat Commun. 2018;9(1):2733
5. Guo Y, Yang H, Chen S, et al. Reduced BAP1 activity prevents ASXL1 truncation-driven myeloid malignancy in vivo. Leukemia. 2018;32(8):1834-1837.
6.  Jadhav SP, Kumari N, Ng L, et al. circASXL1-1 regulates BAP1 deubiquitinase activity in leukemia. Haematologica, 2020; 105 (7): e343 DOI: 10.3324/haematol.2019.225961.

Resistance to anti-cancer treatments is a significant therapeutic challenge and is typically associated with mutations in signaling pathways that are involved in cell proliferation, survival, and tumorigenesis. Genomic analyses of relapsed pediatric acute myeloid leukemia (AML) patients have identified mutations, deletions, and changes in promoter methylation associated with Wnt-β-catenin signaling[1]^,[2]. Mutations in the PI3K-Akt pathway are also frequently associated with treatment resistance in AML and many other cancers[3]^,[4]. Hematopoietic stem cells are particularly sensitive to mutations in these signaling pathways and numerous studies have shown that targeting each of these pathways separately is associated with poor efficacy and the emergence of resistance[5]. AML resistance not only emerges from chemotherapy but can also be seen in response to immunotherapy[6].

Combined therapies are emerging as a strategy to overcome treatment resistance. Numerous clinical trials are examining the efficacy of combining chemotherapy and immunotherapy-based approaches or using differing immunotherapy combinations to enhance anti-tumor immunity. Other studies are seeking to use existing drugs in different ways to avoid resistance. A recent study has shown that the anthracycline antibiotic doxorubicin (DXR) can be repurposed as a β-catenin inhibitor that targets leukemia stem cells (LSCs)[7]. DXR has typically been used as a chemotherapeutic agent at high doses but is associated with significant toxicity. In this study, activation of both the Wnt-β-catenin and PI3K-Akt pathways are associated with the expansion of LSCs. A high throughput screening of a small molecule library identified DXR as an inhibitor of β-catenin activation mediated by interactions with Akt, and low-dose DXR treatment was sufficient to inhibit LSC expansion, even in chemo-resistant pS⁵⁵²-β-cat⁺ LSCs. DXR could also reduce the expression of the immune checkpoint molecules PD-L1, TIM3, and CD24 on LSCs and thus reverse immune checkpoint-mediated resistance. Findings from a pilot clinical trial indicated that low-dose treatment with the DXR analogue daunorubicin of adult patients with relapsed or refractory AML can specifically target chemo-resistant pS⁵⁵²-β-cat⁺ LSCs.

Future studies using next-generation sequencing technology, computational analysis, and high throughput screening will continue to advance the development of treatments for relapsed or refractory cancers. Progress also continues to be made in advancing combined treatments for both hematological malignancies and solid tumors.

1. Hogan, L. E. et al. Integrated genomic analysis of relapsed childhood acute lymphoblastic leukemia reveals therapeutic strategies. Blood. 2011; 118: 5218–5226.

2. Bolouri, H. et al. The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions. Nat. Med. 2018; 24: 103–112.

3. Koren, S. & Bentires-Alj, M. Tackling resistance to PI3K inhibition by targeting the epigenome. Cancer Cell. 2017; 31: 616–618.

4. Lindblad, O. et al. Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML. Oncogene. 2016; 35: 5119–5131.

5. Fruman, D. A. & Rommel, C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev. Drug Discovery. 2014. 13; 140–156.

6. Sharma, P. et al. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. 2017. 168; 707–723.

7. Perry, J.M. et al. Overcoming Wnt-β-catenin dependent anticancer therapy resistance in leukemia stem cells. Nat Cell Biol. 2020. 22(6); 689-700.