Effect of the radioprotector chlorophyllin on graft versus leukemia response in experimental allogeneic hematopoietic stem cell transplantation

Abstract: Total body irradiation (TBI) is an essential component in the conditioning regimens prior to bone marrow transplantation (BMT) for various hematologic malignancies. However, it is associated with acute toxicities such as oral mucositis and pneumonitis that compromise its utility. Chlorophyllin (CHL), a semi-synthetic derivative of chlorophyll is known for its ability to protect against radiation-induced normal tissue damage. Therefore, CHL could be potentially used to overcome radiation-induced complications in the transplant setting. Nevertheless, it is desirable to establish the effect of CHL on the ability of the graft to eradicate residual leukemic cells [graft versus leukemia (GvL)] response. Herein, we developed a GvL model by exposing the BALB/c mice to whole-body irradiation and transplanting leukemic cells (A20) and bone marrow cells from C57BL/6 mice. Our data indicated that CHL did not compromise with the beneficial GvL effect as evidenced by the absence of leukemic signs (hind-leg paralysis) in A20+BMT and A20+BMT+CHL groups. All animals in these groups died of graft versus host disease (GvHD) and survival times were not different between the groups. None of the animals in either group showed clinical (hind-limb paralysis) or histopathological evidence of leukemia in the form of leukemic nodules in the liver. The absence of interference of CHL on the beneficial GvL effect may allow its clinical translation in leukemia patients who require TBI as a conditioning regimen prior to BMT. Encouraged by these results, CHL is being investigated as a radiation-pro.

Introduction

Radiation therapy (RT) is one of the most commonly used treatment modalities across cancers [1]. Even though radiation therapy techniques have evolved, the efficacy is still limited by untoward toxicities that could have life-threatening consequences, especially in the setting of hemopoietic stem cell transplant (HSCT) [2]. The currently used contemporary radioprotectors (amifostine and palifermin) are not always effective and are associated with significant clinical and financial toxicities. Therefore, there is an unmet need to develop novel radioprotectors to overcome the adverse and often morbid reactions of RT [3].

Chlorophyllin (CHL) is a semi-synthetic derivative of chlorophyll-containing mixture of sodium and copper salts [4], and has been shown to have favorable radioprotective effect [5]. Earlier, in the preclinical study, our group demonstrated the radio-protective effect of CHL through hematopoiesis modulation and an increase in stem cell activity [5]. This work led us to initiate several clinical trials to mitigate RT induced toxicities in not only solid tumors like rectal cancer, head and neck cancer, gliomas, and bladder cancers but also in patients undergoing HSCT for hematological malignancies (CTRI/2022/02/040314, CTRI/2023/07/055047, CTRI/2023/08/056133, CTRI/2023/08/056166).

Total body irradiation (TBI) along with high dose chemotherapy is the most commonly used conditioning regimen which allows the homing of donor cells in the bone marrow. It involves irradiating the bone marrow before hematopoietic stem cell transplantation (HSCT) in patients with high-risk and relapsed leukemias [6,7]. The newly transfused hematopoietic stem cells (known as graft) produce lymphocytes that have the ability to identify the residual disease and eradicate it [8]. This beneficial response of the graft over leukemia is known as the graft versus leukemia (GvL) effect [8]. Total body irradiation (TBI) is associated with toxicity of normal organs, which may predispose the recipient to graft versus host disease (GvHD). Therefore, use of radioprotector like CHL is appealing in this context. A prerequisite for any pharmacotherapy to develop in a transplantation setting is that the drug should not compromise the beneficial GvL effect of the graft [9]. Therefore, in the current study, we evaluated the effect of CHL on GvL response.

To investigate the effect of CHL on GvL, 24 female BALB/c mice (6–8 weeks old at the beginning of the experiment) were first randomized into control (n = 12) and CHL (n = 12). After 3 days of CHL treatment, all mice (CHL and control both groups) were exposed to TBI of 6.5 Gy and were further randomized into four groups of six mice each: [1] A20 (from the control group) [2] A20+CHL (from CHL group) [3] A20+BMT (from the control group) [4] A20+BMT+CHL (from CHL group). A20 cells were procured from ATCC (cat: ATCC-TIB-208). Each mouse was injected with 3x10⁶ A20 cells via the tail vein. A-20 cells are B-cell lymphoma cell line which is derived from BALB/c mice. Additionally, bone marrow transplantation was performed in groups 3 and 4 by intravenous injection (via tail vein) of 15x10⁶ splenocytes and 5x10⁶ bone marrow cells obtained from C57BL/6 mice. CHL was administered via oral gavage from day −3 (three days before transplantation) to day +20 (20 days after transplantation) to groups 2 and 4 at a dose of 180 mg/kg. CHL dose was selected as per our previously published report [10].

The GvL effect was monitored according to earlier published methods by our group and others [11,12]. Briefly, leukemic death was defined by the presence of hind leg paralysis and the presence of tumor nodules in the liver. On the other hand, GvHD deaths were characterized by the presence of GvHD symptoms but the absence of hind-leg paralysis and tumor nodules in the liver. Tumor nodules were assessed using hematoxylin and eosin (H and E) evaluation of the liver by a trained pathologist blinded to the group allocation. GvHD symptoms were based on the following six criteria as per our earlier published report: diarrhoea, weight loss, fur texture, activity, posture, and skin integrity [13].

Mice were monitored daily for any sign of leukemia (based on hind-leg paralysis) or aGvHD. The phenotype of leukemia or aGvHD in each group is represented in Fig. 1A. Mice in the A20 alone and A20+CHL group showed signs of leukemia, in contrast, mice in A20+BMT and A20+BMT+CHL groups were devoid of any signs of leukemia (Fig. 1A–B) but died of GvHD. The median survival in the experimental groups was 13.5, 13, 16, and 14.5 days in A20, A20+CHL, A20+BMT, and A20+BMT+CHL groups respectively (Fig. 1C). Cumulatively, these results suggest that CHL does not compromise the GvL effect. At the same time, it does not have an anti-leukemic effect of its own.

Fig. 1.

Effect of CHL on beneficial GvL response: Following radiation, mice were divided into four study groups [1]: A20 [2] A20 + CHL [3] A20 + BMT [4] A20 + BMT + CHL. (A) Representative mouse from each group showing leukemic and aGvHD phenotypes. (B–C) Leukemia-free survival and overall survival were assessed using Kaplan-Meier plots. In Fig. 1B, non-leukemic causes of death were censored (green triangle and inverted purple triangle in A20+BMT and A20+BMT+CHL groups respectively). (D) Liver histopathology showing the presence of tumor nodules in A20 and A20+CHL groups but not in A20+BMT or A20+BMT+CHL groups. GraphPad Prism version 8.0 was used to analyze the data. Time-to-event data comprising overall survival and leukemia free survival was assessed using Kaplan-Meier plots. n = 6 mice/group. Red arrow - tumor nodules; Black arrow - nuclear atypia; yellow arrow – mitosis; green arrow – acute inflammation. H and E magnification 1x and 40x.

A20 cells tend to home into the liver and form tumor nodules. We further performed the histomorphological analysis of the liver from the dead mice to confirm the presence of leukemic deposits in the liver. The histology analysis was done by a trained pathologist, who was blinded for the group assignment. Additionally, we also investigated if GvHD was present by histopathological analysis. The liver of mice in A20 and A20+CHL showed multinodular high-grade malignant tumor deposits composed of large cells with moderate to marked nuclear pleomorphism, brisk mitosis including atypical forms, and scant cytoplasm. A20+BMT and A20+BMT+CHL group did not show any leukemia deposits in the liver but showed signs of aGvHD in the form of acute inflammation in the portal region (Fig. 1D). Thus, histopathological findings also confirm that CHL does not hamper the GvL effect. The statistical analysis between the groups is provided in Table 1A–B. Leukemia-free survival (LFS) and overall survival in Table 1A–B were analyzed using the Mantel-Cox log-rank test.

Table 1.

Comparison of (1A) leukemia-free survival and (1B) overall survival between the groups.

Table 1A: Comparison of leukemia-free survival (LFS) between the groups.
Comparison between groups (LFS)	P value	Hazard Ratio
A20 vs A20+CHL	0.04	0.5 (0.15–1.73)
A20+BMT vs A20+BMT + CHL	0.9	Undefined

Table 1B: Comparison of overall survival (OS) between the groups.
Comparison between groups (OS)	P value	Hazard Ratio
A20 vs A20+CHL	0.04	0.5 (0.15–1.73)
A20+BMT vs A20+BMT + CHL	0.5	0.7 (0.2–2.4)

CHL being a radioprotector can be potentially used in TBI conditioning for HSCT to avoid unwarranted radiation-induced toxicities. However, at the same time, a radioprotector should not interfere with the GvL effect [14]. Herein, we demonstrated that CHL did not compromise the beneficial GvL response. It did not have any anti-leukemic activity. Earlier, our group demonstrated that CHL potentially modulates the immune profile by reducing the reactive oxygen species (ROS) levels, T-cell proliferation and by upregulating the levels of anti-apoptotic genes in murine splenocytes [4]. Furthermore, we reported that prophylactic use of CHL in mice exposed to radiation had an increased level of hematopoietic stem cells and progenitor cells (HSPC) compared to controls, and consequently significant reduction in radiation-induced death [5]. This effect was attributed to the activation of an anti-oxidant and anti-apoptotic defense mechanism by CHL via NRF-2 and NF-κB. Further, CHL was found to be orally bioavailable with a half-life of 2.3 h in a preclinical pharmacokinetics study [5]. We hypothesize that the increased levels of HSPCs induced by CHL may contribute to the anti-leukemic effects of the graft. However, this requires further evaluation in future studies. Based on these strong preclinical data we recently completed a phase 1 clinical trial (CTRI/2018/09/015576) on healthy volunteers wherein CHL was found to be safe up till a dose of 3000 mg/day (unpublished data). A subsequent phase 2 trial (CTRI) demonstrated the effectiveness of CHL in the treatment of RT induced hemorrhagic cystitis in patients with pelvic malignancies (unpublished data). Encouraged by these findings and the ability of CHL to mitigate radiation-induced hematopoietic syndrome, we envisaged an exploratory clinical trial of CHL for the mitigation of TBI induced toxicities in HSCT. The drug doses in ongoing trials were determined by our earlier published preclinical studies [10]. Although in the current study we provided compelling data on CHL's effect on GvL, the study has the following limitations and challenges to address: [1] in the future, variability in patient responses, potential drug interactions, or long-term safety, and effect of CHL on donor cell engraftment (chimerism) shall be defined in ongoing clinical trials, [2] we used qualitative measure of leukemia based on hind-leg paralysis, having quantitative measures such as CD45R and luciferase-tagged cell line could add addition rigor to the study, [3] although CHL does not compromised GvL effect, it could not provide a survival benefit, which requires further investigation in the dose and duration of the treatment. In conclusion, the present study provides preliminary evidence supporting our planned exploratory trials in HSCT, highlighting that CHL does not adversely affect the GvL response.

Data availability statement

Data will be made available upon request to the corresponding author.

Ethical approval statement

This study was approved by the institutional animal ethics committee of the Advanced Centre for Treatment, Research and Education in Cancer (ACTREC) (project no. 24/2023). All animal procedures were carried out in compliance with ARRIVE guidelines and UK Animals (Scientific Procedures) Act 1986 and associated guidelines, and the NIH guide for the care and use of laboratory animals (NIH Publication No. 80-23; revised 1978).

Funding statement

This study was supported by the ACTREC-TMC intramural annual scientific fund (Grant no. 4598).

Declaration of generative AI in scientific writing

We declare no AI or AI assisted tools were used during the preparation of this work.

Author contributions

SKG: Conceptualization, Writing - original draft, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization. SY: Data curation, Investigation, Software, Visualization. DS: Conceptualization, Project administration, Supervision. SKS: Conceptualization, Project administration, Supervision. NK: Conceptualization, Project administration, Supervision, Validation, Investigation, Methodology, Writing - review and editing. JSG: Conceptualization, Project administration, Supervision, Validation, Investigation, Methodology, Writing - review and editing. VG: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - review and editing.

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.