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SALIQ in Rhabdomyosarcoma: A Repurposed Multidrug Regimen to Augment Standard Treatments by Adding Simvastatin, All Trans Retinoic Acid, Lithium, Itraconazole, and Quercetin

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Submitted:

28 September 2023

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28 September 2023

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Abstract
Rhabdomyosarcoma is a cancer arising from arrested myogenic differentiation, seen mainly in children or adolescents. Metastatic rhabdomyosarcoma is often fatal even with aggressive cytotoxic chemotherapies, surgery, and irradiation. SALIQ is an acronym for a multidrug augmentation regimen designed as an adjunct to current rhabdomyosarcoma chemotherapies. SALIQ uses five common non-oncology drugs, repurposed from general medicine use, to promote malignant clone maturation and inhibit rhabdomyosarcoma growth. The five drugs are: the cholesterol lowering drug simvastatin, the acne medicine tretinoin (ATRA), the psychiatric drug lithium carbonate, the antifungal drug itraconazole, and the food supplement quercetin. All five drugs are in common use for non-cancer conditions, are cheap, have an eminently safe side effect profile, and all five have preclinical evidence and good rationale for inhibiting rhabdomyosarcoma growth.
Keywords: 
Subject: Medicine and Pharmacology  -   Oncology and Oncogenics

1. Introduction

This paper introduces SALIQ, a new adjunctive multidrug regimen for treating rhabdomyosarcoma that is projected to impose a low side effect burden. SALIQ is an acronym for the regimen that uses five already marketed non-oncology drugs with well-established data showing that they can inhibit one or another of the known growth drivers active in rhabdomyosarcoma.
Rhabdomyosarcoma is mainly a childhood cancer expressing myogenic markers MyoD, myogenin, and muscle related proteins, myosin heavy chain, skeletal α-actin, and desmin [1,2,3,4]. Four basic categories are recognized - embryonal, alveolar, spindle cell/sclerosing, and pleomorphic [5]. Embryonal (e-rhabdomyosarcoma) and alveolar (a-rhabdomyosarcoma) predominate. Children presenting with metastatic rhabdomyosarcoma have a 3-year event-free survival of 30% and overall survival of 49% after standard current cytotoxic treatments [6]. Regimens from intensive chemotherapy up to high doses with stem-cell support and radiotherapy have been tested over the years to little avail [6,7,8]. Studies like these in widely metastatic rhabdomyosarcoma using intensive chemotherapy imposed significant side effect burdens. Therefore we are pursuing regimens that do not lower quality of life for these children. Examples of lower side effect burden regimens: low-dose continuous cytotoxic chemotherapy (daily without interruption), or non-genotoxic drugs active against specific molecular targets, or repurposed multidrug regimens like SALIQ.
Because rhabdomyosarcoma is a cancer of defective myocyte maturation, an overarching theme of SALIQ development was to find drugs to remove blocks to myoblast maturation in rhabdomyosarcoma cells.
Rhabdomyosarcomas are histologically similar, immunohistochemical stain positive for desmin, myogenin, and MyoD but are otherwise biologically diverse [1,3,4,6]. All forms of rhabdomyosarcoma are diseases of impaired myogenic differentiation [7,8].
The approach of SALIQ was to identify the known abnormalities blocking differentiation in rhabdomyosarcoma then looking at currently marketed drugs that have data that they might be repurposed to inhibit these identified differentiation blocks. Many different forces are active in impeding differentiation of the rhabdomyosarcoma cell. Only some of these are discussed here.
Rhabdomyosarcoma’s molecular classification is evolving [9]. a-rhabdomyosarcoma is more common in adolescents and carries a worse prognosis. Many variables influence 5 year survival after diagnosis - most prominently a worse prognosis if metastases are present. Many different treatment regimens have been reported over that last 2 decades using various combinations of cyclophosphamide, dactinomycin, doxorubicin, ifosfamide, radiotherapy, surgical resection, temozolomide, topotecan, vincristine, vinorelbine, and others. Yet a significant number of children still succumb to the disease.
The SALIQ drugs are: 1) the cholesterol lowering drug simvastatin, 2) all trans retinoic acid (ATRA), a vitamin A metabolite used topically in the treatment of acne, 3) the mood stabilizing drug lithium carbonate, 4) the antifungal drug itraconazole, and 5) the dietary supplement quercetin. Since quercetin is found in high amounts in onions, increased dietary intake of onions and dried onion powder are an additional option. How these drugs might impede rhabdomyosarcoma growth will be presented in section 3 below. Before that, in section 2, three core elements of rhabdomyosarcoma growth relevant to the SALIQ drugs´ actions are reviewed. See Table 1 for quick reference to these.

2. Some elements of rhabdomyosarcoma relevant to SALIQ

2. A. PAX and FOXO1

A fusion product of two transcription factors, PAX (Pax7 or Pax3) and FOXO1 is the result of a common chromosomal translocation found in 80% of a-rhabdomyosarcoma. Degree of PAX-FOXO1 expression is correlated with shorter survival [10]. a-rhabdomyosarcoma without this fusion protein clinically resembles e-rhabdomyosarcoma [11]. Both PAX and FOXO1 have an N-terminal DNA binding and a C-terminal transactivation domain. The PAX-FOXO1 fusion protein is more stable than PAX transcription factor alone. Both PAX3/PAX7 DNA binding and transcriptional activation of the fusion protein is greater than wild-type PAX proteins.The FOXO1 transactivation domain is responsible for interacting with basal transcriptional machinery in this fusion product, abnormally and excessively promoting transcription of PAX binding DNA sites (genes). The PAX fragment is selecting which genes are going to be transcriptionally upregulated by the FOXO1 transactivation domain [1,2]. An unhappy union.
PAX-FOXO1 is resistant to proteolytic degradation, is gene amplified, has increased transcription activity, and increased translation in a-rhabdomyosarcoma. Individually PAX and FOXO1 are retained in cytosol - and hence inactive - after phosphorylation by Akt. The fusion protein PAX-FOXO1 is resistant to Akt phosphorylation, hence transported to nucleus more readily than the individual components would be [2]. Pax-FOXO1 fusion proteins are resistant to Akt activity and therefore predominantly reside in the nucleus. Pax3-FOXO1 and Pax7-FOXO1 exhibit up to 100 fold greater transcriptional activity compared to wild type Pax3 and Pax7 proteins.

2. B. Glycogen synthase kinase 3b

PAX3-FOXO1 enhances glycogen synthase kinase 3beta (GSK3) transcription/translation. GSK3 is a serine-threonine kinase that is activated by its tyrosine-279/216 phosphorylation and inhibited by its phosphorylation on serine 9 [12]. The phosphatase PP2A removes GSK3’s inhibiting serine 9 phosphorylation, reactivating GSK3. PP2A activity is inversely correlated with the level of phosphorylated GSK3 in kainic acid excitotoxic mouse brain [13].
Parenthetical note: Inactive PP2A becomes activated in the presence of several common dopamine receptor 2 inhibiting antipsychotic medicines (haloperidol, paliperidone, penfluridol, perphenazine, thioridazine) [14]. Since both lithium [15,16] and phenothiazine class antipsychotic medicines [17,18] have extensive preclinical databases showing potential cancer growth inhibition, it seems they may work against each other in that role vis a vis GSK3 function. The combination might be best avoided until more is known.
GSK3 inhibition allows non phosphorylated beta-catenin’s nuclear maturation functions to proceed. Beta-catenin phosphorylated by GSK3 is degraded in the proteasome. This process is depicted in Figure 1. See section 5. below on lithium for details.
GSK3 overactivity contributes to the undifferentiated, proliferative phenotype in rhabdomyosarcoma [19].

2. C. MyoD, myogenin, and myogenesis

Myogenin is a gene derepressed by the functioning transcription factor MyoD. Myogenin’s protein product, myogenin, is essential for myogenic differentiation [2,20,21]. Any event blocking or hindering myogenin transcription would be predicted to contribute to maintaining rhabdomyosarcoma [22].
It is probable that e-rhabdomyosarcoma results from an earlier differentiation stage than a-rhabdomyosarcoma because a-rhabdomyosarcomas express three times more myogenin than does e-rhabdomyosarcoma.
MyoD is a muscle specific transcription factor. It can induce a differentiated nonmuscle cell to redifferentiate to a myocyte without going through a pluripotent stem cell stage but cannot do this without myogenin [21]. MyoD and myogenin exist in an amplifying feedback loop maintaining each other’s expression in normal skeletal muscle development and maturation [2,20,21]. Many MyoD cofactors (peptides, proteins, miRNAs, long noncoding RNAs, histone deacetylases, etc) are recognized, and potentially required, that allow or inhibit MyoD to act as a myocyte differentiation promoting transcription factor [23].
Rhabdomyosarcomas express MyoD but preliminary data indicate that these cofactors are preventing MyoD from derepressing maturation programs in rhabdomyosarcoma [2].
Forced ectopic expression of PAX/FOXO1 in murine myoblasts or satellite cells generated malignant cells similar to rhabdomyosarcoma [24]. The PAX/FOXO1 fusion protein fails to derepress myogenin [24].

3. Simvastatin

Simvastatin is a common cholesterol lowering medicine, widely prescribed worldwide. Simvastatin and other cholesterol lowering drugs of this class, the “statins”, are 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors. They have a considerable preclinical database showing potential for cancer growth inhibition [25,26,27,28,29,30]. Clear clinical benefit from statin use has not yet been shown.
Currently marketed examples of statins are atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. Pravastatin and rosuvastatin are hydrophilic, simvastatin and atorvastatin are lipophilic [31,32]. It has been the more lipophilic statins that have shown greater preclinical potential to inhibit cancer growth [25,30].
In adults, high doses of simvastatin (~80 mg/day) carry a higher risk of muscle pain or rhabdomyolysis compared with other statins [33]. This might be a plus as well as a minus when treating rhabdomyosarcoma. This higher muscle pain with simvastatin compared to that seen with other statins may indicate that simvastatin, as a lipophilic statin, attains higher muscle tissue levels than do other statins [34].
HMG-CoA reductase is the rate-limiting step in cholesterol synthesis, converting HMG-CoA to mevalonic acid. Simvastatin is highly lipophilic, metabolized by CYP3A4, and has a T½ of 2 hours although clinical suppression of HMG-CoA reductase is continuous [35].
Oddly, i) despite these preclinical observations of potential benefit of simvastatin, and 2) the generally benign nature of simvastatin, and 3) the serious threat to life from rhabdomyosarcoma, yet as of 2023 there has been no published clinical trial of simvastatin or any other HMG-CoA reductase inhibitor as treatment adjunct in rhabdomyosarcoma.
Proliferation and invasion of mature muscle cells, including those within the venous wall, are inhibited by lipophilic statins atorvastatin or simvastatin but not by hydrophilic statins like pravastatin [36].
HMG-CoA reductase statins also inhibit trans-prenyltransferases including geranylgeranyl pyrophosphate synthase resulting in depletion of geranylgeranyl diphosphate stores in muscle cells and rhabdomyosarcoma growth inhibition [37,38]. Simvastatin inhibits rhabdomyosarcoma cell line growth in vitro by depletion of geranylgeranyl diphosphate [38]. Lovastatin also inhibited in vitro proliferation of rhabdomyosarcoma cells [39].
It remains unproven which of the seven approved and marketed statins would be most effective as rhabdomyosarcoma treatment adjunct. Preclinical evidence would indicate simvastatin.

4. ATRA

ATRA (tretinoin is synonym) is a normal and essential metabolic product of retinoic acid forms (vitamin A) present in our food. ATRA is probably the primary biologically active form of the retinoids. It activates all three main retinoid receptors (RAR): RAR-alpha, RAR-gamma, and RXR. Vitamin A is an essential nutrient as animals cannot synthesize it [40]. ATRA drives differentiation in many different cell lineages and settings [41]. It is also used as a pharmaceutical driver of differentiation in pediatric neuroblastoma and pediatric acute promyelocytic leukemia [42,43,44]. ATRA has been used successfully as a differentiation inducing agent in treating selected pediatric cancers for decades [42,45,46].
Sine oculis homeobox homolog 1 (SIX1) is a transcription factor crucial in embryogenesis. SIX1 is highly expressed in rhabdomyosarcoma. SIX1 is one of the several transcription factors active in maintaining a muscle progenitor-like state in rhabdomyosarcoma. SIX1 loss induces partial differentiation of rhabdomyosarcoma cells and impedes those cells’ growth in vitro [47]. SIX1 is highly overexpressed in rhabdomyosarcoma where it forms part of the myocyte maturation block [47,48,49]. SIX1 is upregulated, forming part of the drive to pathologic growth, invasion, and metastasis across a variety of cancers [50,51,52,53]. ATRA reduced SIX1 expression in a myoblast cell line [54].
mRNA and protein of SIX1 and its transcriptional coactivators EYA1 and EYA2 are elevated in pulmonary fibrosis. The increased SIX1 here is a reparative reactivation on a basis of chronic alveolar damage - a wound repair triggered developmental program reactivation [55].
ATRA slowed proliferation, and resulted in a more differentiated myocyte phenotype in rhabdomyosarcoma cell lines but did not effect survival in a xenograft murine rhabdomyosarcoma model [56,57]. nanoM ATRA in vitro promoted nontransformed myoblast maturation as reflected by increased cell content of myogenin, myosin heavy chain, and myocyte fusion while inhibiting proliferation [58]. ATRA promoted in vitro differentiation of a patient derived a-rhabdomyosarcoma cell line [59]. Many authors using many different rhabdomyosarcoma cell lines have shown that ATRA increased differentiation signs - myotube-like giant cell formation, increased creatinine kinase, reduced proliferation, etc [60,61,62,63]. However not all rhabdomyosarcoma cell lines are equally sensitive to ATRA mediated differentiation or proliferation inhibition [64,65,66].
ATRA increases rhabdomyosarcoma cell lines’ synthesis and secretion of myogenin [67,68]. As discussed in section 2.C. above, myogenin is the sine qua non for muscle cell differentiation.
Rhabdomyosarcoma uses many autocrine growth signaling systems, synthesizing VEGF and expressing VEGF receptor 1 among them. Anti-VEGF monoclonal antibody slows rhabdomyosarcoma growth and forced expression of the PAX-FOXO1 fusion protein into non-malignant muscle cells, induced expression of VEGF receptor 1 [69,70,71]. ATRA decreased rhabdomyosarcoma cell growth and synthesis of VEGF. Adding exogenous VEGF rescued proliferation [71].
In vitro, 3 microM ATRA inhibited the hepatocyte growth factor (HGF) secretion from a glioblastoma cell line [72]. HGF as agonist for its cognate receptor c-Met is discussed below as a growth drive element in rhabdomyosarcoma.
A search for published research showed no clinical trial results of ATRA (tretinoin), isotretinoin, or any other retinoid in rhabdomyosarcoma.

5. Lithium

Lithium, as lithium carbonate, is commonly used in psychiatry to reduce wide mood swings where its primary locus of action is inhibition of GSK3 [73]. Lithium inhibits GSK-3 in a variety of clinical settings and diseases [74,75,76]. Lithium also has an extensive database on its adjunctive use in treating various cancers [77,78,79].
Experience with Li+ use in children is limited. Lithium has a narrow therapeutic index when treating psychiatric problems. In children weighing 20 kg to 30 kg, 600 mg to 1500 mg/day in divided doses would be a typical dose. Lithium plasma levels should be kept around 1 mEq/L Li+ until further experience might dictate other levels best. Severe side effects are seen as plasma levels approach or exceed 2 mEq/L necessitating regular Li+ blood level monitoring.
GSK3 is the central enzyme inhibited by Li+ that results in some inhibition of malignant growth [15,79,80]. As briefly presented in section 2.B. above, GSK3 is a serine/threonine kinase expressed in all mammalian cells. GSK3 is constitutively active and is negatively regulated by phosphorylation on serine-21 and/or serine-9, primarily by Akt, but also through growth factor stimulation of mitogen activated protein kinases (MAPKs), mTOR, protein kinase A (PKA), and protein kinase C (PKC).
Normal human plasma Li+ levels during psychiatric treatment are typically ~ 1mEq. In vitro cytotoxic LD50 to rhabdomyosarcoma cell lines was between 20 and 50 mM LiCl but in vitro colony suppression was 10% of controls at 10 mM LiCl [81]. Since 10 mM LiCl = about 2 mEq/L Li+ we might get only slight Li+ mediated rhabdomyosarcoma growth suppression.
Li+ stabilization of beta-catenin resulting in its enhanced nuclear signaling, induced embryonic stem cell differentiation in hemangioblast-like cells [82]. It is the intent of Li+ use in SALIQ to do the same in rhabdomyosarcoma cells. Post-natal myogenesis and myocyte maturation processes are accelerated by reduction of GSK3 activity [83,84]. A low-therapeutic (0.5 mEq) dose of Li promoted in vitro myoblast fusion and myogenic differentiation in a myoblast cell line [85].
PAX3-FOXO1 enhances GSK3 activity which in turn represses myogenesis activity. GSK3 repressed muscle creatine kinase promoter activity and hyperphosphorylated myogenin, rendering it nonfunctional in driving muscle differentiation in rhabdomyosarcoma cells [19]. Pharmacological inhibition of GSK3 rescues PAX3-FOXO1 repression of myogenesis in rhabdomyosarcoma cells [19]. This chain of events is schematically depicted in Figure 1.
GSK3 action is integral to inhibiting the WNT/β-catenin pathway. as depicted in Figure 1. Wnt signaling activity is a pro-differentiating force in normal muscle [86,87] as well as in rhabdomyosarcoma [7,88,89]. Wnt agonists decreased proliferation and promoted differentiation in a-rhabdomyosarcoma cells [90].
Again, despite the preclinical data showing Li+ induction of rhabdomyosarcoma maturation and growth suppression plus the wealth of clinical experience and ease of Li use in treating psychiatric illnesses, still as of 2023 there have been no published clinical trials of Li+ in rhabdomyosarcoma.

6. Itraconazole, and Hh

Itraconazole is a widely prescribed antifungal drug. Itraconazole is used in the treatment of glioblastoma as part of the CUSP9v3 protocol and is seeing a resurgence of interest in treating other cancers [91,92,93]. Itraconazole has poor solubility and erratic absorption, necessitating taking itraconazole with Coke™, lemon juice, or other low pH drink. Itraconazole is among the strongest CYP3A4 inhibitors in common use. Itraconazole 5 mg/kg/day in divided doses of an oral solution would be a common dose for fungal prophylaxis in children aged 2–18 years undergoing immunosuppressive cytotoxic cancer treatments [94].
Hedgehog signaling (Hh) is one of the crucial signaling systems directing embryogenesis, wound healing, bone marrow homeostasis and other physiological functions [95,96,97]. Itraconazole inhibits Hh function across a wide range of clinical and experimental conditions [92,93,98,99,100,101].
One could reasonably deduce that normal cells either have work around from an Hh signaling block or that itraconazole in human clinical use provides only weak Hh inhibition since even high doses of itraconazole do not result in major organ system failures.
Hh signaling engagement participates in facilitating growth and treatment resistance across a wide variety of cancers [102,103,104,105,106,107]. This is also the case in rhabdomyosarcoma [88,108,109,110,111,112,113]. Multiple interventions are being explored to add Hh signaling inhibition to current rhabdomyosarcoma treatments [110,114].
Hepatocyte growth factor (HGF) signals via its cognate receptor c-Met to stimulate growth, invasion in mature cells and is crucial during morphogenesis. HGF added to cultured rhabdomyosarcoma cells resulted in increased Hh signaling and migration while a phytochemical from ginger plants, zerumbone, decreased that HGF-Hh provoked nondirected motility [115].

7. Quercetin and onions

Quercetin is widely available as a food supplement. It is found in high amounts in a variety of vegetables, the highest amounts in onions [116]. Problems of poor solubility and strong first-pass catabolism have made human studies of quercetin’s anticancer potential difficult [116]. Formulation to improve solubility and absorption are available but are largely of untested effectiveness in achieving these goals. Quercetin is FDA certified as Generally Regarded as Safe (GRAS) and as such unregulated, making pharmacological attributes of quercetin products uncertain. Although multiple quercetin cancer growth inhibiting effects have been shown in preclinical study, none have yet been shown to do so in human disease [117,118,119,120].
A quercetin with lecithin proprietary product as Quercetin Phytosome 500 mg, gave 150 ng/ml plasma level at 6 hours in healthy humans [121]. A second study in healthy humans showed ~200 ng/ml plasma Cmax [122]. If this is typical for quercetin use in humans, blood levels are unlikely to be sufficient for significant rhabdomyosarcoma inhibition. However, worth a try given quercetin’s benignity.
Pannexin 1 expression is decreased in rhabdomyosarcoma, while increasing its levels decreased in vitro growth [123]. Quercetin exposure increased pannexin 1 transcription in rhabdomyosarcoma cells [124].
HGF and its receptor, c-Met’s tyrosine kinase overexpression contributes to suppressing differentiation in rhabdomyosarcoma [125,126,127,128,129]. Quercetin binds to the ATP binding site of c-Met preventing its in vitro activation by HGF [130]. Quercetin suppressed in vitro melanoma migration by preventing HGF activation of c-Met [131].
Quercetin inhibited HGF stimulation of c-Met in a medulloblastoma cell line with an IC50 of 12 microM [132]. Prostate cancer cells exposed to quercetin had reduced c-Met and Akt phosphorylation and a lower IC50 of doxorubicin [133]. In vitro 25 microM quercetin inhibited glioblastoma cell spheroid formation and migration but c-Met inhibition might not have mediated this [134]. Quercetin had an IC50 of 35 microM to HepG2 hepatoma cells [135]. It is questionable whether this microM concentration is clinically achievable.
c-Met receptor is expressed in myogenic precursor cells, and upon signaling stimulation by its ligand, HGF induces normal myogenic precursor stem cells’ proliferation and migration [136]. c-Met mRNA and protein expression was elevated in rhabdomyosarcoma, exogenous HGF increasing motility but not proliferation [137]. HGF/c-Met signaling contributes to rhabdomyosarcoma’s radioresistance [138]. Rhabdomyosarcoma cells can move toward differentiation with inhibition of c-Met signaling [139].
Abnormally increased c-Met is widely expressed in rhabdomyosarcoma [140,141,142]. Upregulation of c-Met activity contributes to the ability to establish rhabdomyosarcoma metastases. c-Met becomes unrepressed by the rhabdomyosarcoma fusion protein PAX-FOXO1, schematically shown in Figure 1 [143,144,145]. Stronger c-Met expression correlates to greater rhabdomyosarcoma tissue invasion [146]. Histologically, tumors with lower c-Met expression were characterized as more mature and differentiated, suggesting that c-Met expression in rhabdomyosarcoma functions as a suppressor of differentiation.
Clinical study NCT03245151 is of a multi-tyrosine kinase inhibitor, lenvatinib, in pediatric cancers including rhabdomyosarcoma. It is underway but results as of this writing in mid-2023 are unknown. Higher HGF/c-Met signaling activity is associated with lenvatinib resistance in adult cancers [147]. This would potentially make quercetin plus lenvatinib a particularly attractive combination for rhabdomyosarcoma. Lenvatinib blood levels, and hence side effects, can be expected to increase in people on strong CYP3A4 inhibitors like itraconazole [148]. These side effects can often be treated by simple dose reduction [148].
Studies in humans show few side effects from up to 2000 mg quercetin/day. Based on that lack, clinical utility might be doubtful but the preclinical studies showing potential quercetin benefits plus the rarity of side effects make trying quercetin reasonable.
Onions contain high levels of quercetin but absorption from that source is uncertain. Increasing onion intake per individual taste would be a harmless intervention.

8. Pharmacological considerations

The estimated rank order of side effect risk of the SALIQ drugs is, lowest to highest, quercetin, simvastatin, itraconazole, lithium carbonate, ATRA. As a conservative approach, treatment initiation should be in that order and at lower doses than their target doses. Quercetin and simvastatin can be started together on day one. ATRA, Li+ and itraconazole are best started one at a time at weekly intervals. Thus after four weeks a patient will be on all five SALIQ drugs at a low starting dose, suggested doses given in Table 3. Then bring doses up to target dose in the same order. As always in these kinds of multidrug regimens, requirement for dose reductions per individual tolerance can be expected.

8. A. Simvastatin

Studies of pediatric simvastatin use in treating hyperlipidemia showed a side effect profile no different from that of placebo [149]. Being a CYP3A4 substrate, simvastatin is technically contraindicated with itraconazole. But given the likely outcome of metastatic rhabdomyosarcoma after several failed chemotherapies, a careful try of the combination with itraconazole could be considered. There are no published reports of simvastatin overdose in children or adults.

8. B. ATRA

Many potential ATRA side effects have been reported, some serious, but fatalities are rare. Alanine aminotransferase or aspartate aminotransferase elevation or headache would be the most common side effects from ATRA [150]. ATRA is strongly teratogenic so birth control must be in place as applicable.

8. C. Lithium carbonate

Li+ is usually given as lithium carbonate. Blood Li+ levels are universally available and must be done regularly during Li+ treatment. Lithium carbonate treatment of children is not common because pediatric onset manic-depression is not common. But we do have experience in pediatric lithium carbonate use that shows good tolerability in pediatric populations [151]. Thyroid function must be monitored due to Li+ potential to inhibit thyroid function.

8. D. Itraconazole

Since itraconazole is only absorbed in low pH conditions, it must be given with an acidic beverage such as lemon juice, Coke™, or similar. Proton pump inhibitors must be avoided [152,153]. Blood levels are readily available as a standard lab test. About 4% of people on chronic itraconazole will develop a reversible elevation of hepatic function transaminases, and 1 in 9000 develop structural liver damage.
Many drugs are formally contraindicated with itraconazole due to itraconazole’s strong CYP3A4 inhibition. Specifically ATRA and simvastatin are metabolized by CYP3A4 so use is formally contraindicated with itraconazole due to potential for increased blood levels. Many of the drugs listed as contraindicated can be safely used at lower doses than usual with appropriate monitoring and dose adjustment as needed. As with most of our current cytotoxic drugs the risk of serious or fatal side effects must be weighed against the potential benefits and the likelihood of death from the cancer. Greatly complicating this decision process is the unknown degree of benefit - if any - from the SALIQ adjuvant regimen. In clinical practice the combination itraconazole together with statins, including simvastatin, is used with appropriate monitoring and dose reductions [153].

8. E. Quercetin

This is predicted to be the most benign of the SALIQ drugs. There are no known side effects in human studies.

8. Discussion and Conclusions

As amply argued elsewhere, many in the oncology community believe that adding one or two drugs will not potently inhibit a currently deadly metastatic tumor like rhabdomyosarcoma [154,155,156,157,158]. We can not expect any one or two drugs added to current cytotoxic chemotherapies to have strong disease inhibiting effects. Aggressively growing tumors have many workarounds to any one or two growth drive blocks. For now, multidrug regimens will be required. We therefore turn to multidrug combination cancer treatments to achieve fractional cell killing or fractional growth inhibition through drug additivity, as in Palmer et al [156]. And as in Kilmister et al “...effective treatment of cancer may require a multi-target strategy with multi-step inhibition of signaling pathways...in lieu of the long-standing pursuit of a ‘silver-bullet’ single-target approach” [158]. And as Lindsey et al say specifically for osteosarcoma, it “continues rapidly modifying its genotype, thus making potential targeted molecular therapeutics increasingly impractical” [159].
SALIQ also follows a military principle of breaking a crucial enemy supply chain in more than one place. Both ATRA and itraconazole inhibit Hh signaling but at two different points along the Hh signaling chain [160].
There exists more growth driving systems in rhabdomyosarcoma than the ones discussed in this paper. Dozens of cofactors interact with the growth driving systems discussed here. Some of these interacting cofactors enhance, others inhibit, function of those systems. SALIQ takes aim at inhibiting some of the more important physiological growth promoting systems. There are, however, others.
The in vitro SALIQ drug levels showing rhabdomyosarcoma growth inhibition or differentiation promotion might be higher than those attainable in humans. This potential problem might be overcome as in the works of Chow et al and of Hu et al. These authors showed that using four different antihypertensive medicines each with different mechanism of action at doses too low to be effective individually, became effective when combined all four in a single tablet [161,162]. Table 4 lists the quarter doses, usual doses, and primary mechanism of action of the four antihypertension medicines combined in the quadpill of Chow et al [161]. Should the same principle hold in rhabdomyosarcoma SALIQ could be effective even if the predicted human tumor tissue levels would be lower than in vitro effective drug levels.
A caveat: when embarking on any untested treatment regimen like SALIQ, meticulous following by the treating physician is essential.
  • Frequent meetings in person with the treating physician for a standard review of systems and targeted physical exam. Weekly is a minimum during the uptitration establishment phase unless experience with a given regimen allows longer evaluation intervals.
  • Frequent blood test monitoring for bone marrow, liver and kidney function.
  • Starting with low doses, adding one drug at a time, reevaluating after several days then increasing dose as tolerated every few days until target dose met or side effects dictate a lower dose.
  • Patients must have 24/7 ready direct access to the treating physician by phone.
  • Careful attention required to assure patient is taking medicines as directed. People often have trouble keeping these multidrug regimens and the frequent dose changes straight.
  • Once stability of dose of all drugs and tolerability established, monthly meetings will suffice.
  • When these conditions cannot be met, use of multidrug regimens like SALIQ or CUSP9v3 is not recommended until wider experiences with them can determine more suited monitoring recommendations.
These seven recommendations all stem from a basic principle of combat, whether it is fencing, chess, go, or actual warfare. General and warfare theorist Carl von Clausewitz (1780-1831) expressed it as “Every plan which enters too much into the detail of the course of the combat is therefore faulty and ruinous, for detail does not depend merely on general grounds, but on other particulars which it is impossible to know beforehand.” General and later President of the USA Dwight D. Eisenhower (1890-1969) said it this way: “In preparing for battle I have always found that plans are useless, but planning is indispensable.”. These military aphorisms express rules for effective practice of medicine too and are particularly applicable for SALIQ use.
The five drugs of SALIQ might not be the ideal drugs to inhibit the relevant growth and treatment resistance pathways active in rhabdomyosarcoma, but these are the drugs we have today in 2023. Given the likely unfavorable outcome once a rhabdomyosarcoma has widely metastasized plus the expected benign nature of adding the SALIQ drugs to standard treatments, the risk/potential benefit is well worth pilot study.
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Figure 1. Schematic showing relationship between pathologic fusion protein PAX-FOXO1 and several intracellular physiological changes consequent to that. See text for details and references. Omitted are many cofactors that interact with the processes shown here, some of which enhance, some of which inhibit a given reaction. Intermediate steps in these reactions are also omitted from the schematic. Dotted arrows indicate processes inhibited by the indicated drug.
Figure 1. Schematic showing relationship between pathologic fusion protein PAX-FOXO1 and several intracellular physiological changes consequent to that. See text for details and references. Omitted are many cofactors that interact with the processes shown here, some of which enhance, some of which inhibit a given reaction. Intermediate steps in these reactions are also omitted from the schematic. Dotted arrows indicate processes inhibited by the indicated drug.
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Table 1. List of some rhabdomyosarcoma cells’ physiological elements relevant to the SALIQ regimen.
Table 1. List of some rhabdomyosarcoma cells’ physiological elements relevant to the SALIQ regimen.
c-MET a tyrosine kinase stimulated by HGF, its transcription enhanced by Pax3-FOXO1
FOXO1 transcription factor directing transcription of target genes
GGD geranylgeranyl diphosphate
GSK3 glycogen synthase kinase 1 beta, a major inhibitor of myogenesis function
HGF hepatocyte growth factor, the cognate ligand of c-Met
Hh Hedgehog signaling
HMG-CoA reductase 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a statin, the target enzyme simvastatin inhibits
MyoD transcription factor for myocyte differentiation
myogenesis transcription factor necessary for myocyte maturation.
Pannexin 1 an ion channel with lower function in rhabdomyosarcoma
PAX paired box transcription factor, PAX3 or PAX7, protein transcription factor
PAX-FOXO1 PAX3-FOXO1 or PAX7-FOXO1, common abnormal fusion protein driver in rhabdomyosarcoma
Table 2. Overview of the SALIQ drugs. APL, acute promyelocytic leukemia; BSA, body surface area; ped, pediatric.
Table 2. Overview of the SALIQ drugs. APL, acute promyelocytic leukemia; BSA, body surface area; ped, pediatric.
drug ped dose comments
simvastatin 10 to 40 mg/d 0.1% rhabdomyolysis risk over 4 years, CYP3A4 metabolism
ATRA 7 mg/m2/BSA bid is 30% of standard ATRA ped APL dose, CYP3A4 metabolism
lithium carbonate 300-600 mg/d little experience with Li+ in children, renal elimination
itraconazole 5 mg/kg/day strong CYP3A4 inhibition
quercetin 1 to 2 g/d potentially ineffective
Table 3. Suggested starting order, doses, and uptitration schedule. * This cannot be predicted due to strong CYP3A4 inhibition by itraconazole. Doses can be titrated by careful monitoring. ** Blood monitoring required, dose adjusted to target blood level of 1 mEq/L. Recommended starting order - quercetin, simvastatin, itraconazole, lithium carbonate, ATRA. Note that simvastatin with itraconazole is contraindicated due to potential for increased simvastatin levels. These doses are for older children and teenagers. Dose reductions to tolerability can be expected.
Table 3. Suggested starting order, doses, and uptitration schedule. * This cannot be predicted due to strong CYP3A4 inhibition by itraconazole. Doses can be titrated by careful monitoring. ** Blood monitoring required, dose adjusted to target blood level of 1 mEq/L. Recommended starting order - quercetin, simvastatin, itraconazole, lithium carbonate, ATRA. Note that simvastatin with itraconazole is contraindicated due to potential for increased simvastatin levels. These doses are for older children and teenagers. Dose reductions to tolerability can be expected.
drug starting dose target dose
simvastatin 10 mg/day 20 to 40 mg/day
ATRA 7 mg/m2/BSA bid cannot be predicted *
lithium carbonate 300 mg/day 600 mg/day **
itraconazole 2.5 mg/kg/day 5 mg/kg/day
quercetin O.5 g/day 2 g/day
Table 4. Data from Chow et al, ref. 161 showing quadpill dose with percent of common human dose in treating hypertension. ARB, angiotensin receptor blocker. MOA = primary mechanism of blood pressure lowering action.
Table 4. Data from Chow et al, ref. 161 showing quadpill dose with percent of common human dose in treating hypertension. ARB, angiotensin receptor blocker. MOA = primary mechanism of blood pressure lowering action.
drug quadpill dose/d (%) Common dose/d MOA
irbesartan 37·5 mg (25%) 150 mg ARB
amlodipine 1·25 mg (25%) 5 mg Ca++ channel blocker
indapamide 0·625 mg (25%) 2.5 mg thiazide diuretic
bisoprolol 2·5 mg (25%) 10 mg beta blocker

Abbreviations

All trans retinoic acid (ATRA); glycogen synthase kinase 3beta (GSK3); hepatocyte growth factor (HGF); 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA); retinoid receptors (RAR); Sine oculis homeobox homolog 1 (SIX1)

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