Using Human ‘Personalized’ Cybrids to Identify Drugs/Agents That Can Regulate Chronic Lymphoblastic Leukemia Mitochondrial Dysfunction

This study uses personalized chronic lymphoblastic leukemia (CLL) cybrid cells to test various drugs/agents designed to improve mitochondrial function and cell longevity. Age-matched control (NL) and CLL cybrids were created. The NL and CLL cybrids were treated with ibrutinib (Ibr-10 μM), mitochondrial-targeted nutraceuticals such as alpha lipoic acid (ALA-1 mM), amla (Aml-300 μg), melatonin (Mel-1 mM), resveratrol (Res-100 μM) alone, or a combination of ibrutinib with nutraceuticals (Ibr + ALA, Ibr + Aml, Ibr + Mel, or Ibr + Res) for 48 h. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide), H2DCFDA(2′,7′ Dichlorodihydrofluorescein diacetate), and JC1 assays were used to measure the cellular metabolism, intracellular ROS levels, and mitochondrial membrane potential (∆ψm), respectively. The expression levels of genes associated with antioxidant enzymes (SOD2, GPX3, and NOX4), apoptosis (BAX and CASP3), and inflammation (IL6, IL-1β, TNFα, and TGFβ) were measured using quantitative real-time PCR (qRT-PCR). CLL cybrids treated with Ibr + ALA, Ibr + Aml, Ibr + Mel, and Ibr + Res had (a) reduced cell survivability, (b) increased ROS production, (c) increased ∆ψm levels, (d) decreased antioxidant gene expression levels, and (e) increased apoptotic and inflammatory genes in CLL cybrids when compared with ibrutinib-alone-treated CLL cybrids. Our findings show that the addition of nutraceuticals makes the CLL cybrids more pro-apoptotic with decreased cell survival compared with CLL cybrids exposed to ibrutinib alone.


Introduction
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western world, accounting for approximately 25% of adult leukemia. The incidence of CLL in the US is 4.5 cases per 100,000 people, and the median age at the diagnosis is 71 years. The incidence of CLL is higher in individuals with family history, of Caucasian ethnicity, and with Agent Orange exposure [1,2]. First-line therapy typically consists of nucleoside analogues (fludarabine) and alkylating agents (cyclophosphamide, chlorambucil, and bendamustine) in combination with monoclonal antibodies directed against CD20 (rituximab) [3]. Targeted agents, including tyrosine kinase inhibitors, such as ibrutinib (BTK-Bruton's tyrosine kinase inhibitor) and idelalisib (PI3Kδ-phosphoinositide-3-kinase inhibitor), along with small molecules such as venetoclax (B-cell lymphoma 2 gene inhibitor), are currently available in the relapse setting [4].

CLL Patient-Derived Cybrid with Dysfunctional Mitochondria Exhibits Resistance to Ibrutinib Treatment
The mitochondria, an essential intracellular signaling core, are emerging as key determinants of a wide range of features central to cancer's initiation, development, and progression, including metabolic reprogramming, metastasis, and chemotherapeutic resistance [24]. First, we assessed if mitochondrial dysfunction was linked to the development of resistance to ibrutinib treatment in this CLL patient. To test this hypothesis, we created patient-derived cybrid cell lines using platelets collected from an age-matched control (NL) individual and a CLL patient who was not responding to ibrutinib treatment. As shown in Figure 1a, treatment with ibrutinib decreased the survival of the NL cybrid in a dosedependent manner, as expected. While the CLL cybrid cells were significantly resistant to ibrutinib treatment compared with the NL cybrid at the 1.5 µM concentration. This finding indicated that mitochondrial dysfunction diminished the response to the ibrutinib treatment by increasing resistance in the CLL patient. A>G in MT-CYB region have been reported in other cancer types, mitochondrial disease, and normal tension glaucoma (NTG) [19][20][21][22][23].

CLL Patient-Derived Cybrid with Dysfunctional Mitochondria Exhibits Resistance to Ibrutinib Treatment
The mitochondria, an essential intracellular signaling core, are emerging as key determinants of a wide range of features central to cancer's initiation, development, and progression, including metabolic reprogramming, metastasis, and chemotherapeutic resistance [24]. First, we assessed if mitochondrial dysfunction was linked to the development of resistance to ibrutinib treatment in this CLL patient. To test this hypothesis, we created patient-derived cybrid cell lines using platelets collected from an age-matched control (NL) individual and a CLL patient who was not responding to ibrutinib treatment. As shown in Figure 1a, treatment with ibrutinib decreased the survival of the NL cybrid in a dose-dependent manner, as expected. While the CLL cybrid cells were significantly resistant to ibrutinib treatment compared with the NL cybrid at the 1.5 µ M concentration. This finding indicated that mitochondrial dysfunction diminished the response to the ibrutinib treatment by increasing resistance in the CLL patient.  (Ibr) in-combination treatment with mitochondriatargeted nutraceuticals, i.e., alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), and resveratrol (Res) {Ibr + ALA; Ibr + Aml; Ibr + Mel; Ibr + Res} on the survival of the cybrid derived from agematched control (NL) and chronic lymphoblastic leukemia (CLL) patient. (a) The effect of ibrutinib treatment on the survival of NL and CLL cybrids using the MTT assay. (b) MTT assay of the effects of Ibr + ALA, Ibr + Aml, Ibr + Mel, and Ibr + Res treatments on the survival of CLL cybrids. One- Figure 1. Effect of individual ibrutinib (Ibr) and (Ibr) in-combination treatment with mitochondriatargeted nutraceuticals, i.e., alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), and resveratrol (Res) {Ibr + ALA; Ibr + Aml; Ibr + Mel; Ibr + Res} on the survival of the cybrid derived from agematched control (NL) and chronic lymphoblastic leukemia (CLL) patient. (a) The effect of ibrutinib treatment on the survival of NL and CLL cybrids using the MTT assay. (b) MTT assay of the effects of Ibr + ALA, Ibr + Aml, Ibr + Mel, and Ibr + Res treatments on the survival of CLL cybrids. One-way ANOVA was used to analyze the data shown as mean SD. ns denotes non-significant, * denotes p-value 0.05; ** 0.01; *** 0.001; **** 0.0001.
Next we asked whether individual treatment with mitochondria-targeting nutraceuticals such as alpha lipoic acid (ALA) [25], amla [26,27], melatonin [28], and resveratrol [29] or treatment in combination with ibrutinib could overcome the resistance in the CLL patient-derived cybrid. In the absence of ibrutinib, we determined the optimal concentratTion of each nutraceutical in NL and CLL patient-derived cybrids required to promote cell viability, and the concentrations of ALA (1 mM), amla (300 µg), melatonin (1 mM), and resveratrol (100 µM) were chosen (Supplementary Figure S1). Then, in the CLL cybrid, we tested the combination treatment of ibrutinib with the optimal concentration of mitochondrialtargeting nutraceuticals. As shown in Figure 1b, when compared with ibrutinib treatment alone, ibrutinib combination treatment with ALA, amla, melatonin, and resveratrol reduced the mean survival rate in the CLL cybrid by 15% (p < 0.0001), 5% (p = 0.095), 8% (p = 0.008), and 12% (p < 0.0001), respectively. The addition of ALA and resveratrol enhanced the response to ibrutinib in CLL cybrids (lower percent survival) compared with the response found in co-cultures of melatonin plus ibrutinib. Amla had no significant effect on the ibrutinib response in the CLL cybrid. Our findings suggest that these mitochondria-targeted nutraceuticals lowered the survivability of the CLL cybrids exposed to ibrutinib. We then tested the hypothesis that combining the ibrutinib with the nutraceuticals may promote the levels of ROS production and apoptosis.
These findings support that in CLL cybrids, the combined treatments of ibrutinib plus ALA, melatonin, or resveratrol have increased ROS production, which may sensitize the CLL cybrids to lower cell survival compared with ibrutinib treatment alone.

Effect of Ibrutinib on the Expression of Antioxidant, Apoptotic, and Inflammatory Genes in the Patient-Derived CLL and NL Cybrids
Antioxidant gene expression: As shown in Figure 4, when cybrids were treated with ibrutinib (10 µM), there was a significant decrease in the relative fold change of SOD2 (0.4-fold change, p < 0.01), GPX3 (0.7-fold change, p < 0.0001), and NOX4 (0.4-fold change, p < 0.0001) in the NL cybrid. In contrast, in the CLL cybrids, treatment with ibrutinib alone caused a significant increase in the relative fold change of SOD2 (1.7-fold change, p < 0.01), GPX3 (1.8-fold change, p < 0.0001), and NOX4 (0.9-fold change, p < 0.0001) expression. These results suggest that the resistance phenomena that developed against ibrutinib treatment in the CLL cybrid may also have been due to the increased expression of antioxidant, which might have diminished the production of ROS as shown in Figure 2e. Data shown as mean ± SD were analyzed by one-way ANOVA test. ns represents non-significant * indicates p-value ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.

Effect of Ibrutinib on the Expression of Antioxidant, Apoptotic, and Inflammatory Genes in the Patient-Derived CLL and NL Cybrids
Antioxidant gene expression: As shown in Figure 4, when cybrids were treated with ibrutinib (10 μM), there was a significant decrease in the relative fold change of SOD2 (0.4fold change, p < 0.01), GPX3 (0.7-fold change, p < 0.0001), and NOX4 (0.4-fold change, p < 0.0001) in the NL cybrid. In contrast, in the CLL cybrids, treatment with ibrutinib alone caused a significant increase in the relative fold change of SOD2 (1.7-fold change, p < 0.01), GPX3 (1.8-fold change, p < 0.0001), and NOX4 (0.9-fold change, p < 0.0001) expression. These results suggest that the resistance phenomena that developed against ibrutinib treatment in the CLL cybrid may also have been due to the increased expression of antioxidant, which might have diminished the production of ROS as shown in Figure 2e. Heat map representation of treatment with ibrutinib (Ibr) on the mRNA expression of genes related to antioxidant, apoptotic, and inflammation using quantitative real-time polymerase chain reaction (qPCR) in the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Data are shown in the form of relative fold change with respect to housekeeping gene HPRT1 with respect to vehicle control and were analyzed by one-way ANOVA test. ns represents non-significant, p-value ** < 0.01; *** < 0.001; **** < 0.0001. Apoptotic gene expression: Next, we wanted to investigate the effect of ibrutinib on the expression of genes related to apoptosis in the CLL and NL cybrids. Treatment with . Heat map representation of treatment with ibrutinib (Ibr) on the mRNA expression of genes related to antioxidant, apoptotic, and inflammation using quantitative real-time polymerase chain reaction (qPCR) in the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Data are shown in the form of relative fold change with respect to housekeeping gene HPRT1 with respect to vehicle control and were analyzed by one-way ANOVA test. ns represents non-significant, p-value ** < 0.01; *** < 0.001; **** < 0.0001. Apoptotic gene expression: Next, we wanted to investigate the effect of ibrutinib on the expression of genes related to apoptosis in the CLL and NL cybrids. Treatment with ibrutinib (10 µM) resulted in a significant increase in the relative fold change of BAX (fold change = 1.7, p < 0.0001) and CASP3 (fold change = 2.2, p < 0.001) in the NL cybrid ( Figure 4). In contrast, ibrutinib treatment of the CLL cybrid caused a significant decrease in the relative fold change of BAX (fold change = −0.4, p < 0.0001) and CASP3 (fold change = −0.5, p < 0.01) expression ( Figure 4).
Inflammatory gene expression: Treatment of NL cybrids with ibrutinib resulted in a significant increase in the mean fold change of IL-6 (1.3-fold change, p < 0.01) but no change in the expression of IL1β, while there was a significant decrease in the mean fold change of TNFα (0.6-fold change, p < 0.001) and TGFβ (0.7-fold change, p < 0.01). In contrast, ibrutinib treatment of the CLL cybrid caused a significant decrease in the mean-difference fold change of IL1β (p < 0.0001), whereas it had no significant effect on the expression of other inflammatory genes ( Figure 4).

Figure 5.
Heat map representation of treatment with alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), and resveratrol (Res) on the mRNA expression of genes related to antioxidant, apoptotic, and inflammation using quantitative real-time polymerase chain reaction (qPCR) in the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Data are shown in the form of relative fold change with respect to housekeeping gene HPRT1 with respect to vehicle control and were analyzed by one-way ANOVA test. ns represents non-significant * indicates p-value ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.
Interestingly, there were increases and decreases in the expression levels of IL1β in both the NL and CLL cybrids. The significant expression levels of IL1β increased in response to ALA (3.5-fold change, p < 0.0001) and melatonin (1.2-fold change, p < 0.0001) in the NL cybrid, while treatment with amla (0.7-fold change, p < 0.0001) and resveratrol (0.4-fold change, p < 0.0001) significantly decreased the expression levels of IL1β in the NL cybrid ( Figure 5). Similarly, in the CLL cybrid the ALA (3.2-fold change, p < 0.0001) significantly increased the expression levels of IL1β, whereas the other treatments significantly reduced the levels: amla (0.3-fold change, p < 0.0001), melatonin (0.8-fold change, p < 0.0001), and resveratrol (0.5-fold change, p < 0.0001) ( Figure 5).
In the NL cybrids, we observed that supplement-alone treatment with ALA (0.6-fold change, p < 0.0001) and melatonin (0.9-fold change, p < 0.05) had significant decreases in the expression levels of TNFα, whereas there were significant increases in the expression levels of TNFα levels in response to treatment with amla (2.9-fold change, p < 0.0001) and resveratrol (2.3-fold change, p < 0.0001). In the CLL cybrids in response to melatonin (0.7-fold change, p < 0.0001), there were significant decreases in the expression levels of the TNFα levels. In contrast, in response to ALA (4.7-fold change, p < 0.0001), amla (6.1-fold change, p < 0.0001), and resveratrol (1.7-fold change, p < 0.0001), there were increased expression levels of TNFα ( Figure 5).
Next, we found significant increases of TGFβ levels in response to amla (2.1-fold change, p < 0.0001) in the NL cybrid and (three-fold change, p < 0.0001) and in the CLL cybrid ( Figure 5). In contrast, in response to ALA, the NL cybrids showed decreased levels by 0.7-fold change (p < 0.0001), and the CLL cybrid had a 0.5-fold change (p < 0.0001) in the expression levels of TGFβ. NL cybrids showed decreased levels of TGFβ in response to melatonin (0.9-fold change, p < 0.0001) and resveratrol (0.8-fold change, p < 0.0001). In contrast, the CLL cybrids showed a significant increase in TGFβ in response to melatonin (1.2-fold change, p < 0.0001) and resveratrol (1.1-fold change, p < 0.0001) ( Figure 5).

Effect of in-Combination Treatment of Ibrutinib with Mitochondrial Targeted Nutraceuticals on the Expression of Antioxidant, Apoptotic, and Inflammatory Genes in the Patient-Derived CLL and NL Cybrids
Antioxidant gene expression: Next, we wanted to investigate the effect of combining mitochondria-targeting nutraceuticals (ALA, amla, melatonin, and resveratrol) with ibrutinib on the expression of genes related to antioxidants ( Figure 6). In the NL cybrid, Ibr + Amla (Ibr + Amla, 0.4-fold change, p < 0.001) and Ibr + Resv (Ibr + Resv, 0.7-fold change, p < 0.05) significantly decreased the expression of SOD2, while there was no significant change in the expression levels of SOD2 in response to Ibr + ALA (Ibr + ALA) and Ibr + Mel (Ibr + Mel). The CLL cybrid also had lower expression of SOD2 after exposure to the combinations Ibr + ALA (0.2-fold change, p < 0.001), Ibr + Amla (0.9-fold change, p < 0.001), and Ibr + Mel (0.7-fold change, p < 0.05). There was no significant change in the expression of SOD2 in response to treatment to Ibr + Resv (1.2-fold change, ns).

Effect of in-Combination Treatment of Ibrutinib with Mitochondrial Targeted Nutraceuticals on the Expression of Antioxidant, Apoptotic, and Inflammatory Genes in the Patient-Derived CLL and NL Cybrids
Antioxidant gene expression: Next, we wanted to investigate the effect of combining mitochondria-targeting nutraceuticals (ALA, amla, melatonin, and resveratrol) with ibrutinib on the expression of genes related to antioxidants ( Figure 6). In the NL cybrid, Ibr + Amla (Ibr + Amla, 0.4-fold change, p < 0.001) and Ibr + Resv (Ibr + Resv, 0.7-fold change, p < 0.05) significantly decreased the expression of SOD2, while there was no significant change in the expression levels of SOD2 in response to Ibr + ALA (Ibr + ALA) and Ibr + Mel (Ibr + Mel). The CLL cybrid also had lower expression of SOD2 after exposure to the combinations Ibr + ALA (0.2-fold change, p < 0.001), Ibr + Amla (0.9-fold change, p < 0.001), and Ibr + Mel (0.7-fold change, p < 0.05). There was no significant change in the expression of SOD2 in response to treatment to Ibr + Resv (1.2-fold change, ns). Figure 6. Heat map representation of treatment with alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), and resveratrol (Res) on the mRNA expression of genes related to antioxidant, apoptotic, and inflammation using quantitative real-time polymerase chain reaction (qPCR) in the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Data are shown in the form of relative fold change with respect to housekeeping gene HPRT1 with respect to vehicle control and were analyzed by one-way ANOVA test. ns represents not significant * indicates p-value ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.
In the NL cybrid cells, Ibr + Amla (0.7-fold change, p < 0.0001) and Ibr + Mel (0.6-fold change, p < 0.0001) significantly decreased the expression of GPX3, while the Ibr + ALA (1.1-fold change, p < 0.01) treatment significantly increased the expression of GPX3 levels. There was no change in the GPX3 levels after the Ibr + Resv treatment ( Figure 6). In the Figure 6. Heat map representation of treatment with alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), and resveratrol (Res) on the mRNA expression of genes related to antioxidant, apoptotic, and inflammation using quantitative real-time polymerase chain reaction (qPCR) in the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Data are shown in the form of relative fold change with respect to housekeeping gene HPRT1 with respect to vehicle control and were analyzed by one-way ANOVA test. ns represents not significant * indicates p-value ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.
Interestingly, the expression levels of IL1β were significantly decreased in response to Ibr + ALA (0.4-fold change, p < 0.0001) in the NL cybrid, while in response to Ibr + Amla, Ibr + Mel, and Ibr + Resv, there were no changes in the expression levels of IL1β in the NL cybrid ( Figure 6). In contrast, the CLL cybrid showed significantly increased expression levels of IL1β in response to Ibr + Amla (two-fold change, p < 0.0001), Ibr + Mel (3.9-fold change, p < 0.0001), and Ibr + Resv (1.4-fold change, p < 0.0001). There was no significant change in the expression of IL1β in response to Ibr + ALA in the CLL cybrid ( Figure 6).

Discussion
In this study, we generated cybrid cell lines from a CLL patient and an age-matched control subject. Using these transmitochondrial cybrids, we demonstrated that cells containing the CLL mitochondria were more resistant to ibrutinib treatment than cells with age-matched control mitochondria. However, as shown in Figure 3, ibrutinib in combination with ALA, melatonin, and resveratrol (mitochondria-targeted nutraceuticals) significantly sensitized the cells to the ibrutinib, making the percent survival lower than the ibrutinib-alone-treated cells. Moreover, the combination of ALA, melatonin, and resveratrol with ibrutinib increased cellular ROS production, mitochondrial membrane potential, and expression of apoptotic genes and decreased the expression of antioxidant genes in the CLL cybrids but not in the NL cybrids (Figure 7).

Discussion
In this study, we generated cybrid cell lines from a CLL patient and an age-matched control subject. Using these transmitochondrial cybrids, we demonstrated that cells containing the CLL mitochondria were more resistant to ibrutinib treatment than cells with age-matched control mitochondria. However, as shown in Figure 3, ibrutinib in combination with ALA, melatonin, and resveratrol (mitochondria-targeted nutraceuticals) significantly sensitized the cells to the ibrutinib, making the percent survival lower than the ibrutinib-alone-treated cells. Moreover, the combination of ALA, melatonin, and resveratrol with ibrutinib increased cellular ROS production, mitochondrial membrane potential, and expression of apoptotic genes and decreased the expression of antioxidant genes in the CLL cybrids but not in the NL cybrids (Figure 7). Our study showed that the mtDNA from the CLL patient's cybrid was defined as the N haplogroup, which is an ancestral line that descended directly from haplogroup L3. Early members of this group lived in the eastern Mediterranean and western Asia [34]. Mutations in the D-loop region have been reported in human neurodegenerative and Our study showed that the mtDNA from the CLL patient's cybrid was defined as the N haplogroup, which is an ancestral line that descended directly from haplogroup L3. Early members of this group lived in the eastern Mediterranean and western Asia [34]. Mutations in the D-loop region have been reported in human neurodegenerative and ophthalmologic disorders, cardiovascular diseases, and cancers including leukemic. Some researchers identified associations between SNPs m.15784T>C, m.16185C>T, and m.16399A>G SNPs located in the mtDNA D-loop region that have been reported in breast cancer [35,36]. The m.73A>G, m.152T>C, m.189A>G, m.16223C>T, and m.16390G>A SNPs located in the mtDNA D-loop region have been reported in breast cancer [35,36]. The present study identified two private SNPS in the CLL cybrid's mtDNA regions: m.2866A>T (no rs#, RNR2) and m.13635T>C (no rs#, MT-ND5), which were not linked to any known pathogenesis.
A review of the literature shows that pathways involved with the mitochondria are inextricably linked to chemoresistance, which is a survival strategy employed by cancer cells in response to apoptotic stimulation [37]. Cancer cells rely heavily on mitochondria for a variety of functions, including metabolism, calcium signaling, mitochondrial dynamics regulation, and resistance to oxidative stress [37]. Mitochondrial plasticity is involved in many stages of tumor development, including chemoresistance. In fact, recent research suggests that chemoresistant ovarian cancer cells gain an advantage over their non-resistant counterparts when their mitochondria undergo a bioenergetic switch to oxidative metabolism at the same time [38]. Our findings that CLL mitochondria are resistant to ibrutinib treatment while the NL cybrids are sensitive are in line with these studies.
While it is not always clear what causes oxidative stress in cancer patients, it has been recognized that excessive levels of ROS can be produced by tumor cells and cells that interact with tumors. Compared with normal B cells, CLL cells have higher mitochondrial respiration rates, consistent with elevated levels of mitochondria-derived ROS. This, in turn, can cause increased oxidative stress, which can make CLL cells resistant to chemotherapy [39]. Interestingly, in NL cybrid cells, the ibrutinib treatment in combination with ALA, amla, melatonin, and resveratrol did not significantly change the mean difference levels of ROS production compared with the vehicle-treated cells (Figure 7) and led to lower levels of ROS when compared with ibrutinib treatment alone. In contrast the same treatment in the CLL cybrid significantly increased the mean difference levels of ROS by 21% (Ibr + ALA), 35% (Ibr + Mel), and 21% (Ibr + Res), compared with the ibrutinib-alone-treated CLL cells. One can speculate that the CLL cybrid combination treatment produces more ROS than the ibrutinib treatment alone, which may sensitize the CLL cybrid to accelerated cell death.
Diminished mitochondrial membrane potential (∆Ψm) is a hallmark of mitochondrial dysfunction [40]. It has been observed that cancer cells have a more negative net transmembrane potential (∆Ψm) compared with normal cells, which may be caused by alterations in mitochondrial bioenergetics [41]. Our findings show that combining Ibr + ALA, melatonin, and resveratrol improves the mitochondrial health, as evidenced by higher ∆Ψm levels compared with the CLL cells treated with ibrutinib alone. This was surprising because lower ∆Ψm levels are often associated with elevated apoptosis, but our gene expression studies showed that the CLL cybrid treated with the combination of ibrutinib plus ALA, melatonin, and resveratrol had two-fold or greater levels of BAX and CASP3, which are pro-apoptosis genes. As a result of higher mitochondria membrane potential, there may be improved oxidative phosphorylation, which would lead to significantly increased levels of ROS production. This may result in CLL cybrid sensitization to ibrutinib in combination with ALA, melatonin, and resveratrol rather than individual ibrutinib treatment as seen in Figure 3.
The hypersensitivity of normal cells to ROS makes them vulnerable to carcinogenic effects if they are not properly protected by antioxidant mechanisms. In contrast, chemoresistant cancer cells have antioxidant mechanisms (glutathione, SOD, catalase, and others) that are up-regulated, protecting them from ROS [42]. Our data imply that elevated ex-pression of SOD2 and GPX3, as seen in the CLL cybrids ( Figure 6), may be reducing ROS production and contributing to the CLL cybrid resistance to ibrutinib treatment.
Targeting BAX proteins and permeabilizing the mitochondrial outer membrane induces apoptosis in chemotherapy. Pro-apoptogenic substances from the mitochondrial intermembrane space are released into the cytosol by mitochondrial outer membrane permeabilization, activating a caspase cascade that kills the cancer cell [43,44]. Our results raise the possibility that the decreased expression of apoptotic genes, BAX (0.4-fold) and CASP3 (0.5-fold), contributes to CLL cybrids' resistance to ibrutinib treatment by increasing cell survival. Conversely, the expression of apoptotic genes was not as greatly affected by treatment with mitochondria-specific nutraceuticals alone (range 0.8-fold to 1.1-fold change). In contrast, ibrutinib treatment with ALA, melatonin, and resveratrol significantly up-regulates the expression of BAX (range 2-fold to 3.4-fold) and CASP3 (2-fold to 3.9-fold) in CLL cybrid cells.
Some of the negative effects of chemotherapy include an increase in pro-inflammatory cytokines [45,46], which can contribute to cancer-related fatigue and pro-inflammatory cytokines that play a pivotal role in chemotherapy-related side effects [47]. Many chemotherapeutics, such as cisplatin, etoposide, ibrutinib, paclitaxel, topotecan, and vinblastine, cause cell death by inducing DNA damage, which causes nuclear and mitochondrial DNA leakage into the cytosol and activates cGAS-STING signaling, which is part of innate immunity [48,49]. Our findings show that in-combination treatment up-regulates the proinflammatory genes in CL cybrids to higher levels than seen in the NL age-matched control cybrid cells. This would result in more inflammation and untoward side effects in the CLL patients compared with what might be found in cells with normal mitochondria.
Our results show that using mitochondrial-targeted nutraceuticals with ibrutinib increased cellular ROS production, mitochondrial membrane potential, and expression of apoptotic genes and decreased the expression of antioxidant genes in CLL cybrids. These results suggest that mitochondria are critical players in cancer cell survival because they serve as the bioenergetic and biosynthetic hub that coordinates cellular respiration, ETC, apoptosis, antioxidant signaling, and redox homeostasis. Cancer drug resistance, as an adaptive strategy used by cancer cells to survive stress conditions, is inextricably linked to mitochondrial-related pathways. Indeed, emerging evidence strongly suggests that resistant tumor cells have high mitochondrial respiration and OXPHOS status. As a result, targeting mitochondria represents a promising cancer treatment avenue and chemoresistance-overcoming strategy.

Generation of Patient-Derived Cybrid
Transmitochondrial cybrids were created by fusing a mitochondrial DNA-deficient ARPE-19 (Rho0) cell line with platelets isolated from either chronic lymphoblastic leukemia (CLL) patients or age-matched control, normal (NL) subjects ( Figure 8). Ten milliliters of peripheral blood were collected in tubes containing 3.2% sodium citrate from age-matched NL and CLL patients. To isolate platelets, blood was centrifuged, and final platelet pellets were resuspended in Tris-buffered saline. ARPE-19 cells Rho0 cells (mitochondria DNAdeficient) were created by passaging ARPE-19 cells in low doses (50 ng/mL) of ethidium bromide several times and (0.025 g) uridine supplement in the media. The Rho0 cells were fused with platelets via polyethylene glycol fusion, and the newly formed cybrids were then cultured to passage 5 in media containing DMEM-F12, 10% dialyzed fetal bovine serum, 100 unit/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL fungizone, 50 µg/mL gentamicin, and 17.5 mM glucose.

Whole mtDNA Genome Sequencing of the CLL Cybrid
The sequencing method was a modified version of one used by Zaragoza et al. [50]. We were able to quantify the SNPs that are homoplasmic, heteroplasmic, haplogroup defining, private (non-haplogroup defining), and unique (those not listed in www.Mito-Map.org) across the entire mitochondrial genome of the CLL subject using this sequencing method. The PCR for whole mtDNA genome sequencing was performed in two parts using a high-fidelity PCR system (FailSafe TM PCR System, Lucigen, Madison, WI) and two sets of primers. Part A used primers hmtL569 [AACCAAACCCCAAAGACAC] and hmtH12111 [AAACCCGGTAATGAT-GTCGG], while Part B used primers hmtL11727 [GCCCACGGGCTTACATC] and hmtH1405 [ATCCACCTTCGACCCTTAAG] (Integrated DNA Technologies, Inc., Coralville, IA). The PCR products were run on a 1% agarose gel, and the unincorporated primers and dNTPs were eliminated enzymatically in a single step (ExoSAP-IT, Thermo Fisher Scientific, Pittsburgh, PA, USA). Samples were sent to ELIM BioPharm for sequencing using internal primers (ELIM Biopharm, Hayward, CA, USA). The sequencing results were analyzed using DNA variant analysis software (Mutation Surveyor, SoftGenetics, State College, PA, USA). The mtDNA sequences were compared with the classification from www.hmtvar.uniba.it (accessed on 12 October 2022).

Identification of mtDNA Haplogroup in the CLL Cybrid
The DNA was extracted from cell pellets from the CLL patient-derived cybrids using the Purelink TM Genomic DNA Mini Kit (Invitrogen, Thermo Fisher Scientific, Carlsbad,

Whole mtDNA Genome Sequencing of the CLL Cybrid
The sequencing method was a modified version of one used by Zaragoza et al. [50]. We were able to quantify the SNPs that are homoplasmic, heteroplasmic, haplogroup defining, private (non-haplogroup defining), and unique (those not listed in www.MitoMap.org) across the entire mitochondrial genome of the CLL subject using this sequencing method. The PCR for whole mtDNA genome sequencing was performed in two parts using a high-fidelity PCR system (FailSafe TM PCR System, Lucigen, Madison, WI) and two sets of primers. Part A used primers hmtL569 [AACCAAACCCCAAAGACAC] and hmtH12111 [AAACCCGGTAATGAT-GTCGG], while Part B used primers hmtL11727 [GCCCACGGGCTTACATC] and hmtH1405 [ATCCACCTTCGACCCTTAAG] (Integrated DNA Technologies, Inc., Coralville, IA). The PCR products were run on a 1% agarose gel, and the unincorporated primers and dNTPs were eliminated enzymatically in a single step (ExoSAP-IT, Thermo Fisher Scientific, Pittsburgh, PA, USA). Samples were sent to ELIM BioPharm for sequencing using internal primers (ELIM Biopharm, Hayward, CA, USA). The sequencing results were analyzed using DNA variant analysis software (Mutation Surveyor, SoftGenetics, State College, PA, USA). The mtDNA sequences were compared with the classification from www.hmtvar.uniba.it (accessed on 12 October 2022).

Identification of mtDNA Haplogroup in the CLL Cybrid
The DNA was extracted from cell pellets from the CLL patient-derived cybrids using the Purelink TM Genomic DNA Mini Kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), according to the manufacturer's instructions. Mitochondrial DNA profiles were determined through sequencing, as has been previously described [51]. In general, the mtDNA haplogroup profile for the cybrids was compared with the sequence of the blood samples to verify the successful introduction of the mitochondria with the personalized mtDNA into the cybrid cell line.

Cell Survival
NL and CLL cybrids were plated in a 96-well plate at a density of 10,000 cells per well in 100 µL of culture media. After 24 h plating, the cells were treated for 48 h with ibrutinib, ALA, amla, melatonin, and resveratrol at concentrations of 10 µM, 1 mM, 300 µg, 1 mM, and 100 µM, respectively. After the completion of the treatment, the cell metabolism was measured using the colorimetric MTT assay kit according to the manufacturer's protocol (MTT Cell Viability Assay Kit, Biotium, Fremont, CA, USA). A minimal of three replicates were tested per sample. Each experiment was repeated three times.

Reactive Oxygen Species (ROS) Assay
The NL and CLL cybrids were plated in a 96-well plate at a density of 10,000 cells per well in 100 µL of culture media. Twenty-four hours after plating, the cells were treated for 48 h with ibrutinib, ALA, amla, melatonin, resveratrol, and in combination at concentrations of 10 µM, 1 mM, 300 µg, 1 mM, and 100 µM, respectively. Following the treatment process, the cells were exposed to the fluorescent H 2 DCFDA (2,7-dichlorodihydrofluorescin diacetate) (Invitrogen-Molecular Probes, Carlsbad, CA, USA) dye. The ROS levels were measured using a fluorescence plate reader (Gemini XPS Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 492 nm and emission wavelength of 520 nm. A minimum of three replicates were tested per sample. Each experiment was repeated three times.

JC-1 Mitochondrial Membrane Potential (∆Ψm) Assay
The NL and CLL cybrids were plated in a 96-well plate at a density of 10,000 cells per well in 100 µL of culture media. After 24 h plating, the cells were treated for 48 h with ibrutinib, ALA, amla, melatonin, resveratrol, and in combination at concentrations of 10 µM, 1 mM, 300 µg, 1 mM, and 100 µM, respectively. Following the treatment process, the cells were exposed to 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanineiodide (Biotium, Hayward, CA, USA) for 15 min. The mitochondrial membrane potential was then measured with a fluorescence plate reader (Gemini XPS Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) by measuring the fluorescence for red (excitation 550 nm and emission 600 nm) and green (excitation 485 nm and emission 545 nm). Intact mitochondria with normal ∆Ψm appeared red, whereas impaired cells with diminished ∆Ψm were green. The ratio of red/green was used for analysis. A lower ratio corresponded to higher apoptotic/dead cell number. A minimum of three replicates were tested per sample. Each experiment was repeated three times.

RNA Extraction, cDNA Synthesis, and qRT-PCR
Patient-derived NL and CLL cybrids were cultured and incubated for 24 h in sixwell plates. Twenty-four hours after plating, the cells were treated 48 h with ibrutinib, ALA, amla, melatonin, resveratrol, and in combination at concentrations of 10 µM, 1 mM, 300 µg, 1 mM, and 100 µM, respectively. After the completion of 48 h of treatment, RNA extraction was performed on both the NL and CLL cybrid cells using an RNA isolation kit (Purelink TM RNA Kit, Ambion, Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer's protocol. The RNA quantity and purity were measured on a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). An OD 260/280 of 1.8-2.0 indicated the purity of RNA devoid of protein contamination. Concentrations of 500 ng/µL RNA were reverse transcribed into cDNA using a SuperScript TM VILO TM IV cDNA Synthesis Kit (Thermo Fisher Scientific). The mRNA levels of mitochondrial biogenesis genes were determined by quantitative reverse transcription-PCR (qRT-PCR) using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). Reactions were carried out in a final volume . Each PCR reaction was followed by continuous melt curve analysis. No template controls (NTCs) were also included in each PCR run to assess contamination. All samples were run in triplicate. Data analyses were performed after normalization to the reference gene to calculate a fold change value using the ∆∆Ct method, which was calculated by subtracting the difference between the threshold cycles (Cts) of the target gene and the housekeeper gene (HPRT1 gene). The fold change was calculated using the following formula: fold change = 2 −∆∆Ct . A fold change value ≥ 1 was considered as 'up-regulation', whereas a fold change value < 1 was considered as 'down-regulation' of the gene.

Statistical Analysis
GraphPad Prism 9 software was used to perform statistical analyses on all quantitative data (GraphPad Software, Inc., San Diego, CA, USA). All of the analyses were compared with the vehicle control group. Unless otherwise specified, comparisons between experimental groups were made using one-way ANOVA followed by Sidak's multiple comparisons method.

Conclusions
To conclude, mitochondria from CLL patients are more likely to become resistant to ibrutinib treatment than those from age-matched control patients while ibrutinib combined with mitochondria-targeted nutraceuticals, especially ALA, melatonin, and resveratrol, significantly sensitizes CLL cybrids by increasing the cellular reactive oxygen species (ROS) production, mitochondrial membrane potential, and expression of apoptotic genes and decreasing the expression of antioxidant genes.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement:
In terms of various phases of this study including designing, performing, and revealing of the results, patients or the public were not involved.

Data Availability Statement:
All data relevant to the study are included in the article.

Conflicts of Interest:
All authors declare that they have no competing interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.