Autoimunitate versus apoptoză la unele cazuri de leucemie limfocitară cronică

Introduction

Chronic lymphocytic leukemia (CLL) is frequently associated with immune disturbances. The aim of this study was to emphasize the mechanisms leading to autoimmune cytopenia in CLL and to examine the interactions between malignant B-CLL cells and abnormally functioning B cells within the cellular microenvironment (Figure 1).

Apoptosis is characterized by specific pathways initiated by the activation of death receptors (DRs), followed by a downstream signaling cascade involving the mitochondria, subsequent caspase activation and DNA cleavage. Proapoptotic signals, such as the TNF-related apoptosis-inducing ligand (TRAIL), tumor necrosis factor (TNF) and the Fas receptor (also known as APO-1 or CD95), act as key proteins that transmit apoptotic signals mediated by the primary DRs. These signals participate in a cascade that culminates in the cleavage of a specific set of proteins, ultimately resulting in the disassembly of the cell⁽¹⁾.

While some autoreactive cells may escape natural apoptosis and persist despite treatment, potentially leading to an autoimmune response, apoptotic cells do not simply disappear after phagocytosis. Components of these cells can survive intracellular processing and be recycled to the phagocyte membrane. Consequently, massive apoptosis that overloads phagocytic capacity may trigger an autoimmune reaction through the presentation of nucleosomes to the immune system⁽²⁾.

In recent years, extensive studies have examined various mature B-cell malignancies, including CLL, focusing on p53 protein isoforms produced by mutant TP53 genes in hematologic malignancies involving the 17p chromosomal deletion. Identifying these mutations is critical, as they significantly impact the clinical course of patients with CLL expressing mutant p53 isoforms.

The mitochondria provide an ideal molecular platform for the counter-regulation of autophagic versus apoptotic cell death. In this regard, mitochondria-associated proteins may also facilitate interactions between autophagic and apoptotic pathways. Furthermore, mitochondria play a central role in inducing apoptosis by releasing cytochrome C following the disruption of the mitochondrial outer membrane potential (Scheme 1).

The frequency of gene mutations, deletions or translocations of the TP53 gene in CLL can be classified as biomarkers of the individual proteomic and genomic profiles for this type of leukemia.

Changes in microRNA expression and aberrant methylation patterns in genes specifically dysregulated in CLL, including BCL-2, TCL1 and ZAP-70, have also been identified and linked to distinct clinical parameters. Specific chromosomal abnormalities and genetic mutations may serve as diagnostic and prognostic indicators for disease progression and survival. Consequently, the efficacy of new therapeutics should be evaluated based on the presence of these molecular lesions in CLL patients.

Apoptosis and autoimmunity

Apoptotic gene activity is regulated by the wild-type TP53 gene. Its product, the p53 protein, inhibits the nuclear factor-kappa B (NF-kB) signaling pathway, which is responsible for protein synthesis during inflammation in autoimmune diseases. Mutations in the TP53 gene lead to a loss of p53 protein function, which can trigger autoimmune processes⁽³⁾.

In this context, autoimmunity serves as an interface for malignancy, with the TP53 gene acting as a central element. The insufficient action of the p53 protein due to genetic mutations facilitates this transition toward autoimmunity⁽³⁾.

Furthermore, autoreactive B and T cells that escape natural apoptosis may represent an additional necessary condition for disease progression. The link between apoptosis and tumor necrosis factor (TNF) activity explains why abnormal TNF production plays a critical role in several autoimmune diseases; supplemental mutations in these pathways may ultimately drive these cells toward oncogenesis⁽⁴⁾.

Apoptosis and malignancy

The primary regulatory mechanisms in both autoimmunity and oncogenesis include death receptors (DRs), caspases and the mitochondria, as well as the Bcl-2 family of proapoptotic proto-oncogenes, such as Bak and Bax, and the tumor suppressor gene TP53. Treatment with venetoclax, a potent inhibitor of the antiapoptotic Bcl-2 membrane receptor, works by neutralizing overexpressed Bcl-2. This inhibition triggers the initiation of the mitochondrial (intrinsic) apoptotic pathway (Scheme 2).

Mitochondrial activities leading to apoptosis include the disruption of cellular membrane transport and the loss of mitochondrial membrane potential, impacting the cellular reduction-oxidation (redox) balance. This revised terminology accurately reflects the biological mechanisms involved in CLL cell death.

In a healthy cell, the p53 nuclear protein binds to DNA and stimulates the expression of the CDKN1A gene. This gene produces the p21 protein, which interacts with a cell division-stimulating protein, cyclin-dependent ki­nase 2 (CDK2)⁽⁵⁾.

The expression of CDKN1A is tightly regulated by tumor suppressor protein p53, which mediates cell cycle arrest at the G1 phase in response to various stressors. When the p21 protein forms a complex with CDK2, the cell is prevented from transitioning to the next stage of division, the G1-S phase. In a normal cell, p53 is kept inactive by its negative regulator, MDM2. Upon DNA damage or other cellular stress, specific signaling pathways trigger the dissociation of the p53-MDM2 complex. Once activated, p53 induces cell cycle arrest to allow for either DNA repair and cell survival or apoptosis to eliminate the damaged cell⁽⁵⁾ (Scheme 3).

Methylation of the CDKN2A gene (INK4a/ARF locus) can epigenetically silence the expression of the p14-ARF protein, thereby blocking the ability of activated oncogenes to stabilize the p53 response. In experimental models, disrupting the MDM2-p53 interaction restored the p53 function and sensitized tumors to chemotherapy or radiotherapy. This strategy could be particularly beneficial in treating cancers that lack TP53 mutations or other specific genetic lesions (such as 11q, 13q, 13-14q or 11q22-q23 deletions; 7q21-q23 deletions; or trisomy 12)⁽⁶⁾.

For example, this approach may be effective in hematologic malignancies such as multiple myeloma (MM) and chronic lymphocytic leukemia (CLL), specifically when the 17p chromosomal arm and the TP53 gene remain intact, as well as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin’s disease and non-Hodgkin lymphoma.

In cases involving TP53 mutations, immunotherapy using p53 anti-peptide antibodies is currently being tes­ted. In these tumor types, the induction of p53 through small-molecule MDM2 inhibitors, such as Nutlin, can successfully induce apoptosis in malignant cells.

The most vital regulatory mechanisms in autoimmunity and oncogenesis include death receptors, caspases, mitochondria, the Bcl-2 proto-oncogene family and the p53 tumor suppressor gene (Scheme 4)⁽⁷⁾.

The p21 protein, a regulator of cell cycle progression from the G1 to S phase, is controlled by the tumor suppressor p53. Consequently, an increased frequency of MDM2 gene amplification is observed in many human cancers; this serves as a mechanism to downregulate p53 activity through ubiquitin-dependent proteasomal degradation of intracytoplasmic p53. A mutant p53 protein can no longer bind effectively to DNA, and, as a result, its downstream effector, p21, is unavailable to serve as the “stop signal” for uncontrolled cell division (Scheme 5).

The p21 protein, which regulates cell cycle progression from the G1 to the S phase, is controlled by the tumor suppressor p53. This mechanism is based on findings reported in high-concentration anaerobic ATP studies in aborted apoptosis from CLL⁽⁸⁾.

Antibodies specific for total p53 and for reactive p53, phosphorylated at three distinct sites within the activation domain, were used in parallel analyses across various malignant diseases. The substitution of the serine-15 amino acid in the p53 protein with alanine results in a partial failure of p53 to inhibit cell cycle progression, thereby driving oncogenesis. In this context, nuclear p53 has been shown to protect the cell from malignant transformation. In contrast, cytoplasmic p53 isoforms phosphorylated at multiple sites, within a modified cytoplasmic environment characterized by high concentrations of anaerobic ATP, facilitate the development of cancerous diseases.

In most tumors, the p53 protein is inactivated by TP53 gene mutations that lead to the production of a mutant protein with increased stability in B-cell lymphocytes. This stability allows for the identification and quantification of the mutant p53 protein using various molecular techniques, such as immunohistochemistry, PCR, SNP analysis or ELISA. This is in contrast to normal B cells, which contain wild-type p53, a protein present only in small amounts due to its very short half-life⁽⁸⁾.

The expression of unmutated immunoglobulin heavy chain variable (IGHV) genes, ZAP-70 and CD38 proteins, along with chromosomal abnormalities such as 17p deletions are associated with a poor prognosis in CLL. Furthermore, mutations in tumor suppressor genes, such as TP53 and ATM, are linked to resistance to conventional chemotherapeutic agents⁽⁹⁾.

Changes in microRNA expression and aberrant methyl­ation patterns in genes specifically dysregulated in CLL, including BCL-2, TCL1 and ZAP-70, have also been reported in the literature. These molecular changes are linked to clinical parameters that distinguish various stages of disease progression and relapse in CLL.

Specific chromosomal abnormalities and genetic mutations serve as diagnostic and prognostic indicators for disease progression and survival in patients with CLL. Consequently, the efficacy of new therapeutics should be evaluated based on the presence of these molecular lesions.

Frequent alterations in the P53 gene are found in more than 75% of CLL cases. MDM2 overexpression leads to the repression of numerous p53-dependent genes and mRNAs, including microRNA-34a (miR-34a)⁽¹¹⁾.

Alterations in microRNA expression and aberrant methylation patterns in genes specifically dysregulated in CLL, including BCL-2, TCL1 and ZAP-70, have also been identified and linked to distinct clinical parameters. Since this microRNA is involved in inducing apoptosis and cell cycle arrest, a more aggressive disease course may correlate with the dysregulation of miR-34a.

A large cohort study of primary CLL examined samples from over 100 patients to assess their response to MDM2 inhibition. The study found a direct correlation between wild-type p53 status and the ability of MDM2 inhibitors, such as Nutlin-3 and MI-219, to induce cytotoxicity across various CLL subtypes. This response was not predicted by other clinical biomarkers, such as ZAP-70 or CD38 expression, unmutated IGHV genes or mono-allelic ATM gene loss. Additionally, many cancer patients produce p53-reactive phosphorylated T cells; for instance, over 40% of breast cancer patients have p53-reactive CD4+ and CD8+ T cells in their peripheral blood. These responses occur most frequently in patients with high p53 expression in their tumors⁽¹²⁾.

Current research indicates that p21 levels are strongly correlated with mammalian target of rapamycin (mTOR) activity. Furthermore, adenosine monophosphate-activa­ted protein kinase (AMPK), which induces the acetylation, methylation and phosphorylation of p53, activates the p53 protein in response to DNA damage (Scheme 6).

Intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance in human cancer cells across various genetic backgrounds. Drug-resistant cells are characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of anaerobic intracellular ATP and enhanced HIF-1a-mediated signaling. When cellular ATP concentrations are high (typically >5 mM), it is thought that higher drug concentrations are required for the efficacy of ATP-competitive inhibitors, posing potential toxicity risks⁽¹³⁾.

In a study published in the February 2, 2016 online edition of Nature Communications, researchers noted that, through the Warburg effect, glucose maintains the stability of mutant p53 and promotes cancer cell survival. Most research indicates that, in line with its role as a tumor suppressor, p53 is capable of inhibiting glycolysis⁽¹⁴⁾. The mTORC2/Akt complex controls mitochondrial metabolism and physiology by phosphorylating the glycolytic enzyme hexokinase 2, thereby promoting aerobic glycolysis (the Warburg effect) and preventing mitochondrial apoptosis⁽¹⁵⁾.

Furthermore, Aurora kinases (A and B) play critical roles in regulating spindle assembly, chromosome segregation and cytokinesis to ensure the faithful segregation of chromosomes during mitosis. Conversely, the aberrant expression of Aurora kinases causes defects in mitotic spindle assembly, checkpoint activation and chromosome segregation, leading to chromosomal instability. While Aurora-A affects p53 activity and stability, Aurora-B phosphorylation of p53 at Ser269 and Thr284 inhibits p53 transactivation activity. Additionally, phosphorylation at Ser183, Thr211 and Ser215 accelerates p53 degradation via the MDM2 protease pathway⁽¹⁶⁾.

Autoimmune phenomena are frequently observed in CLL patients, and are primarily attributable to underlying immune system dysfunction. Autoimmune cytopenia (AIC) afflicts 4-7% of CLL patients, primarily presenting as autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP). The clinical definition of AIHA requires the following criteria: hemoglobin (Hb) levels ≤11 g/dL in the absence of cytotoxic treatment in the preceding month or other identified etiologies; evidence of an underlying autoimmune mechanism, such as a positive direct antiglobulin test (DAT) for IgG or com­ple­ment C3 (or the presence of cold agglutinins), la­bo­ratory markers of hemolysis, such as elevated reti­cu­locyte counts, low serum haptoglobin and increased levels of serum lactate dehydrogenase (LDH) and indirect bilirubin⁽¹⁷⁾.

While the association between CLL and autoimmune cytopenias like AIHA and ITP is well established, there is no definitive proof of an increased risk for non-hemic autoimmune disorders in CLL. The mechanisms leading to AIC in CLL are complex, involving interactions between malignant B-CLL cells, dysfunctional T cells, the microenvironment and the broader immune system. Patients presenting with anemia associated with anti-erythrocyte autoantibodies (Ae-Ab) are diagnosed with CLL-associated AIHA. The Ae-Ab complex and complement bound to the red cell membrane are detected by DAT using a broad-spectrum antiserum⁽¹⁸⁾.

AIHA occurs in 5% to 10% of CLL patients, while ITP occurs in 2% to 5%. AIC may be diagnosed prior to, at the time of, or at any point during the course of CLL, in both treated and untreated patients. Furthermore, AIC tends to appear in high-risk CLL cases (e.g., unmutated IGHV, or 17p and 11q deletions)⁽¹⁹⁾.

Studies conducted in 2025 show that highly effective CLL treatments combining fludarabine with other agents (e.g., FCR) are associated with a lower incidence of AIHA compared to previous standard chemotherapies. Taken together, these results suggest that a lack of treatment response, rather than the treatment itself, conveys a higher risk of AIC. Additionally, retrospective studies have confirmed the efficacy of ibrutinib and idelalisib in managing AIC when present in CLL patients⁽²⁰⁾.

Conclusions

The research presented here has a direct impact on clinical patient management and necessitates a shift toward tailored therapeutic strategies within the framework of personalized medicine. By integrating advanced diagnostic tools, comprehensive knowledge databases and targeted therapies, personalized medicine can be effectively applied to improve patient outcomes.   

Abbreviations: AMPK 5’-AMP: activated protein kinase;HIF-1a: hypoxia-inducible factor-1a; MAP: kinase interacting kinase; MAPK: mitogen-activated protein kinases; MEK: MAPK/ERK kinase; mTOR: mammalian target of rapamycin; MDM2: mouse double minute 2 homolog; PI3K: phosphatidyl ino­sitol-4,5-bisphosphate-3-Kinase; TGF-a: transforming growth factor a; VEGF: vascular endothelial growth factor

Corresponding author: Aurelian Udriştioiu E-mail: aurelianu2007@yahoo.com

Conflict of interest: none declared.

Financial support: none declared.

This work is permanently accessible online free of charge and published under the CC-BY licence.