Insights into existing and futuristic treatment approach for chronic myeloid leukaemia

Treatment

CML treatment relies on the disease phase at the time of diagnosis. Also, one patient can receive either only one type of therapy or a combination of therapies. Initially, therapeutic modalities for CML encompassed chemotherapeutic agents and immunomodulatory approaches; however, since the advent of tyrosine kinase inhibitors (TKIs) in 2000 for targeted therapy, this class of pharmaceuticals has substantially supplanted conventional treatment strategies.

Targeted therapy

In CML, TKI drugs target the BCR-ABL kinase enzyme. There are only five TKIs accessible for treating CML which are divided as first and second line treatment.

First-line treatment and second-line treatment

First-line treatment comprises TKIs for newly identified CML patients. Presently, only four TKIs are finalised under firstline treatment against CML. This includes first-generation drug imatinib and second-generation drugs (2GTKIs) like dasatinib, nilotinib and bosutinib. All these TKIs have similar working mechanisms. These drugs bind to ATP binding sites on the kinase domain of BCR-ABL oncoprotein and then obstruct the transfer of the phosphate group to the protein substrate and its consecutive stimulation. As a result, proliferative signals are blocked, and apoptosis is induced in leukaemic cells. The details of all these TKIs are mentioned in Table II^13-18. Firstline treatment is shifted to secondline treatment after its failure, i.e. resistance or intolerance of initial TKI. The sole TKI which cannot be used in firstline treatment and is developed only for secondline treatment is ponatinib. Failure of ponatinib after three months of treatment leads to the recommendation of early allogeneic-stem cell transplantation (Allo-SCT).

Treatment beyond second-line: stem cell transplantation (SCT)

An insignificant reaction to two or more TKIs leads to considering SCT. The objective of SCT is to eradicate cancer cells in the body either by chemotherapy or by radiation and then substitute the bone marrow containing leukaemic cells with blood-forming HSCs. Out of autogenic and allogenic, exclusively allogenic-SCT (Allo-SCT) is used to heal CML in which healthy blood stem cells are taken from another person of the same species and then transplanted into the diseased patient. In advanced CML, second or third-generation TKIs are introduced to bring down CML load and then recommended for premature Allo-SCT^19,20. If primary 2GTKI prescribed under first or second-line treatment fails, the patient is recommended Ponatinib or an experimental agent. Simultaneously, the patient is evaluated for Allo-SCT and a donor hunt begins. Patients in CML-AP should be considered high-risk patients and should proceed to SCT without a lag as progression-free survival (PFS) in the blast phase is extremely low.

Molecular monitoring & treatment-free remission (TFR)

Molecular monitoring of BCR-ABL1 transcripts is used to evaluate the response of TKIs in CML patients. It is used to check the treatment failure and to alter the therapy timely. Although the clear-cut BCR-ABL1 transcript level and duration of the maintenance period after which treatment termination can be tried have not yet been decided, it is expected that the results of clinical trials on the TKI termination study will be out in the coming years. Presently, major molecular response (MMR, BCR-ABL1 ≤ 0.1% on International Standard (IS)) is an ideal response. Further deep molecular response (DMR) is a new frontier in CML treatment. DMR can be described as BCR-ABL1 levels of molecular response (MR)⁴ (BCR-ABL ≤ 0.01%) and molecular response (MR)⁴^,⁵ (BCR-ABL ≤ 0.0032%) on the IS²¹. Non-success in obtaining MMR by the 12 months is related to a low rate of deep response²². Hence, molecular monitoring is an essential element in managing patients with CML and the TFR of recovered patients.

Treatment-free remission in medical terms refers to a decrease in or disappearance of signs and symptoms of cancer for a specific period. However, only 40-60 per cent of patients showed accomplishment in attaining TFR²³. The first investigation on the concept of ending TKI treatment was through the Stop Imatinib 1 (STIM1) study²⁴ in which 100 patients who attained complete molecular response (CMR) after more than two years of IM treatment were terminated from TKI therapy. Further, 42 patients (61%) experienced molecular relapse after TKI therapy termination, while 26 patients again attained CMR after IM reintroduction. Whereas, after stopping nilotinib and dasatinib, the chance of upholding TFR has been analogous to the results after stopping IM (approximately 50%)^25,26. More extended periods of TKI therapy, DMR and former treatment with IFN-α were spotted as crucial prognostic factors for TFR success in the EURO-SKI trial²⁷, the largest trial of TKI discontinuation After support from ENESTfreedom (192 wks, phase 2 trial)²⁸ and the ENESTop (192 wks) study, Nilotinib (NIL) has obtained approval from the health authorities to support TFR^28,29. In ENESTfreedom and ENESTop studies, the TFR rate measured at the end of the study was 44.2 and 46.0 per cent, respectively. High TFR rates can be accomplished with 2GTKIs due to intense and more sustained MR attained with 2GTKI than IM.

Given that 50-60 per cent of patients with undetectable DMR are anticipated to lose MMR, real-time qPCR may not be the best method for molecular monitoring during TFR^30,31. Therefore, enhanced detection methods are needed to boost the perfection in detecting BCR-ABL1 transcripts and further aid in picking up the patients suitable for TFR. In recent years, digital PCR (dPCR) has completely changed how MRD in haematological diseases is molecularly monitored³². In dPCR, the biological sample is divided into numerous distinct reactions, each undergoing more efficient amplification in microscopic partitions. Partition positivity or negativity depends on template molecule presence, and Poisson’s statistics are applied to determine nucleic acid copies in the initial sample. In this manner, a more sensitive absolute quantification can be carried out without the use of an external calibration or standard curve³³. dPCR can detect one BCR-ABL1 positive cell in 10⁷ cells and is less susceptible to non-specific amplification and inhibitory agents that can damage DNA³⁴. While dPCR offers advantages and applications, it has limitations, such as longer experimental durations and susceptibility to errors in pre-analytical stages like sampling, RNA extraction followed by cDNA synthesis. When analyzing PB samples directly, the Cepheid GeneXpert system, a cartridge-based automated real-time qPCR technique, can be used to find BCR-ABL1 p210 transcripts. The GeneXpert instrument combines RNA extraction, RT-PCR and BCR-ABL1 fluorescence detection in one reaction by employing microfluidics in a cartridge³⁵. Therefore, this instrument offers the advantage of a quick and easy-to-use method, requiring little technical knowledge. But for an accurate estimation of BCR-ABL1 on the IS, it is imperative to create and use a specific conversion factor (CF), along with calibration specific to the cartridge lot³⁶.

Continuous burden of CML

Massive research has been done in the field of CML, and several treatment modalities and diagnoses have been developed; still, it is a rising global burden. There are two main factors responsible for the loss of response and continuous global burden in CML, i.e. BCR-ABL1 independent and BCR-ABL1 dependent mechanisms of resistance.

Drug transporters

Drug transporter proteins mediate drug movement, with balanced TKI influx and efflux crucial for BCR-ABL1 inhibition, while an imbalance contributes to TKI resistance in CML. The human organic cation transporter 1 (OCT1), the membrane influx pump, is highly responsible for IM uptake inside the cell and the ATP-binding cassette (ABC) efflux pump is used for IM movement outside the cells³⁷. Low OCT1 expression is a typical feature of multidrug resistance and is associated with sub-optimal responses. While OCT1 functional activity in leukaemic cells at diagnosis can predict TKI response, the relationship between OCT1 expression and IM transport or response remains controversial, with many studies showing no significant connection^38,39. Other transporters, including OCTN2, OATPs and MATE1, are mediators of TKI transport. According to Alves et al.’s observation, OCT1 and OCTN2 expression decreased simultaneously, demonstrating the involvement of multiple influx transporters in the resistance process⁴⁰. Similarly, overexpression of the ABCB1 gene that encodes P-glycoprotein (P-gp), one of the ABC efflux transporters, could decrease intracellular IM levels, reducing therapeutic efficacy⁴¹. The ABCG2 gene encodes the breast cancer resistance protein (BCRP), a key TKI resistance transporter, especially relevant in protecting LSCs⁴². Reduced drug influx or increased efflux may foster BCR-ABL1 mutations in CML cells and other resistance mechanisms.

Signalling pathways and regulatory factors

In response to BCR-ABL1 inhibition, CML cells can activate alternative signalling pathways like JAK/STAT, PI3K/AKT, RAS/MAPK and SRC, enabling proliferation and survival despite effective BCR-ABL1 suppression⁴³. JAK2 activation by cytokines from cancer and bone marrow niche cells phosphorylates STAT members, including STAT5, which promotes CML development by enhancing the cell cycle and ROS production, inhibiting apoptosis and increasing P-gp expression⁴⁴. Upon PI3K activation, AKT undergoes phosphorylation and affects several downstream proteins. AKT targets include BAD, where it suppresses apoptotic signal. Additionally, AKT phosphorylates transcription factors FOXO, blocking their activity, thus preventing apoptosis and promoting the cell cycle⁴⁵. Ma et al⁴⁶ identified increased RAS/MAPK pathway activity contributing to IM resistance in CML-LSCs. Elevated activity of the protein kinase C (PKC) family within this pathway promotes leukaemia cell proliferation and inhibits apoptosis even without BCR-ABL1 kinase activity. Overexpression of SRC family members like LYN and HCK has been associated with TKI resistance in CML³⁷. These SRC proteins trigger STAT5 activation to promote proliferation and AKT activation to promote survival. Utilising alternative signalling pathways alongside BCR-ABL1 drugs promises to enhance drug response and prevent resistance in CML treatment.

Epigenetic alterations

The BCR-ABL1 mutation not only causes the HSC to become an LSC but also triggers epigenetic reprogramming. The epigenetic processes are generally categorised into three main groups: histones and their modifications, DNA methylation and non-coding RNAs.

Post-translational modifications on histone tails, such as methylation or acetylation, alter chromatin structure and recruit factors. Dysregulated histone-marking systems in CML impact leukaemic cell survival pathways. Polycomb-group (PcG) proteins, including PRC1 and PRC2, are epigenetic regulators dysregulated in CML LSC. Elevated EZH2 in CP-CML LSCs perform tri-methylation of histone H3 on lysine 27 (H3K27me3) at PRC2 target genes, altering CML LSCs survival dependence on EZH2⁴⁷. BMI1, a crucial factor in PRC1 activity, exhibits elevated levels in CD34+ cells with CP-CML and correlates with disease severity. Increased BMI1 expression is associated with decreased CCNG2 (cyclin G2) expression, which inhibits autophagy. Histone variant, γ-H2AX, at DNA damage sites, mediates critical cellular decisions for DNA repair or apoptosis, with dysregulation evident in CML⁴⁸. In CD34+ CML cells, SIRT1, a NAD-dependent HDAC, is upregulated and has been linked to the survival of LSCs by carrying out deacetylation on a number of targets including p53⁴⁹. The protein arginine methyltransferases (PRMT) family of HMTs can either activate or inhibit transcription through the methylation of histone arginine. It is believed that PRMT5 plays a crucial role in the epigenetic regulation of canonical Wnt signalling, a pathway that is crucial for LSC function⁵⁰. Inhibiting PRMT5 activity in CML CD34+ cells decreased LSC numbers invitro⁵¹. HDAC inhibitors like Panobinostat, MAKV and Chidamide with IM demonstrated synergistic anticancer responses and increased therapeutic effectiveness in CML cells⁵².

Transcription of genes is inhibited by DNA methylation. Proliferation and motility of CML LSC and progenitor cells are likely to be increased by MTSS1 (a tumour suppressor) repression but can be decreased by MTSS1 enforced expression⁵³. Durable MTSS1 induction was achieved in CML cell lines following in vitro treatment through the demethylating agent 5-azacytidine. The apoptotic activator BCL2-like protein (BIM) has been demonstrated to undergo epigenetic reprogramming after TKI therapy and downregulated BIM levels are linked to decreased optimal reactions⁵⁴. IM and the demethylating agent 5-aza-deoxycytidine together increased the expression of BIM and reduced the viability and cell proliferation of CML cell lines⁵⁴. Short strands of non-coding RNAs (ncRNAs) called microRNAs (miRNAs) frequently bind to the 3’ UTR of a target mRNA, and either stop translation or cleave the mRNA⁵⁵. miR-150, miR-146a and miR-10a expression were significantly lower at CML diagnosis when compared to healthy individuals; however, following a short period of IM therapy, their expression levels returned to normal^56-58. The expression profiles of several microRNAs, including the miR-17-92 cluster, also known as oncomir-1, differ in CML. At the time of diagnosis, CD34+ cells from CML patients had higher expression levels of oncomir-1⁵⁹. Within 14 days of beginning IM therapy, Flamant et al. discovered a reduction in oncomir-1 expression in MNC⁵⁸.

BCR-ABL1 kinase domain (KD) mutation

A point mutation in the KD of BCR-ABL oncoprotein changes its configuration due to which TKIs can no longer attach to it effectively. As the chemical structure and mechanism of action of each TKI drug are slightly different, one TKI may be able to overcome the resistance from a mutation that another TKI cannot. Mutations are responsible for generating resistance in nearly two-thirds of resistant CML-AP and CML-BP patients and around one-third of resistant CML-CP patients.

T315I gatekeeper mutation, identified as the first BCR-ABL KD mutation, provides resistance against first and 2GTKIs. However, Dasatinib helped to overcome most Imatinib-resistant mutations but was not able to overcome the T315I mutation⁶⁰. This mutation can be treated with drugs like Ponatinib and Synribo (Omacetaxine). Other resistant point mutations are Y253F, F317L, G250E, M351T and V256G, and two novel frameshift mutations are Glu281 and Tyr393. All these mutations affect the P-loop, gatekeeper, activation and catalytic loop domain region of BCR-ABL protein and cause poor Imatinib binding in the ATP binding region⁶¹.

There are plenty of clinical trials ongoing for treating CML under different types of mutations, drug resistance, and checking the efficacy of new or combinations of drugs. Some of the registered clinical trials (Supplementary Table) and drugs are as follows:

Drugs under clinical trial

The role of TKIs has a fixed position in first-line treatment; therefore, most of the trials focus on TFR or TKI holidays. A distinct category of clinical trials in CML is researching developing treatments behind relapse, with their main concern on Ponatinib and Bosutinib. Other encouraging trials involve a combination of TKI with various agents that target non-BCR-ABL1 proteins. Additionally, clinical trials are going on for third-line (3L) treatment where new CML therapies targeting BCR-ABL1 are in development, including drugs like PF114, HQ1351 and Asciminib.

Future hope to cure CML: CRISPR/Cas

To overcome the issue of the production of more oncoproteins from unaltered BCR-ABL1 oncogene, genome editing tools can be used as new alternative therapeutics. The opportunity to knock down oncogenes is now practical with the emergence of genome-editing nucleases like transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas). Among various genome editing tools, CRISPR/Cas is under extensive research for treating genetic disorders.

Research work on CRISPR/Cas9 in the field of leukaemia has immensely accelerated in the past few years^64-68. The evolution of mouse models that imitate human CML⁶⁹ has granted new possibilities to assess the therapeutic function of CRISPR/Cas9. CRISPR/Cas9 tool perhaps rectifies the acquired mutations of the human leukaemia cell line⁷⁰. In 2016, the foremost clinical trials engaging CRISPR/Cas9 work in humans began⁷¹. It was reported in 2017 that the CRISPR/Cas9 system could successfully abolish the BCR-ABL1 oncogene⁶⁴. Nullification of BCR-ABL1 oncogene by another genome editing nuclease, ZFNs, was demonstrated in 2018⁶⁵. Recently, it was reported that the human CML cell line (K562) displayed a diminishing proliferation rate after the use of the CRISPR/Cas9 lentiviral vector to obstruct ABL1 in BCR-ABL1 oncogene⁷², which has exposed the therapeutic capability of CRISPR/Cas system. CRISPR/Cas 9 functioned as the therapeutic system by obstructing the BCR-ABL1 oncogene by aiming at ABL1 exon 2⁶⁷, fusion sequence⁶⁸ and ABL1 exon 6⁷³ while ZFNs obstructed the BCR exon 1⁶⁵.

This genome editing tool still possesses some technical limitations, but the number of available substitutes to conquer them has accelerated at the same rate. A reliable method of delivery is the significant limitation of in-vivo CRISPR/Cas therapy. The CRISPR toolkit can be delivered as Cas9 mRNA and gRNA or as plasmid DNA⁷⁴. Popular viral vectors for in-vivo CRISPR component delivery include Adeno-associated-virus (AAV), Lentivirus (LV) and Adeno-virus (AdV). Despite their high in vivo transfection efficiency, concerns about clinical use remain, such as immunogenicity and integration⁷⁵. Another concern is the pre-existing adaptive immunity against Cas9 in humans, necessitating the use of new Cas proteins. Off-target effects (OTEs) are common when using CRISPR/Cas9 for gene therapy⁷⁴. Another limitation of the technology is the requirement for a nearby PAM sequence. Streptococcus pyogenes Cas9 (SpCas9), a commonly used variant, detects a short PAM sequence (5′NGG3′). However, its size challenges gene therapy delivery via AAV vectors. To broaden the gene target range, various SpCas9 variants have emerged, like SpCas9-NG and xCas9^76,77. Even though CRISPR editing in humans is still a hotly debated and contentious subject, a few FDA-approved and RAC-reviewed CRISPR gene therapy trials have begun after careful analysis of the risk-to-benefit ratios. These initial approved trials, which are now in Phase I/II, are only for patients with severe illnesses, like cancers or incapacitating monogenic diseases.

In the future, it is expected that the CRISPR/Cas9 therapy will turn out to be routine clinical practice.