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.