Complex I Inhibitor of Oxidative Phosphorylation in Advanced Solid Tumors and Acute Myeloid Leukemia: Phase I Trials

Abstract

Although targeting oxidative phosphorylation (OXPHOS) is a rational anticancer strategy, clinical benefit with OXPHOS inhibitors has yet to be achieved. Here, we advanced IACS-010759, a highly potent and selective small-molecule complex I inhibitor, into two dose-escalation phase I trials in patients with relapsed/refractory acute myeloid leukemia (NCT02882321, n=17) and advanced solid tumors (NCT03291938, n=23), respectively. The primary endpoints were safety, tolerability, maximum tolerated dose, and recommended phase 2 dose (RP2D) of IACS-010759. The PK, PD, and preliminary antitumor activities of IACS-010759 in patients were also evaluated as secondary endpoints in both clinical trials. IACS-010759 had a narrow therapeutic index with emergent dose-limiting toxicities, including elevated blood lactate and neurotoxicity, which obstructed efforts to maintain target exposure. Consequently, no RP2D was established, only modest target inhibition and limited antitumor activity were observed at tolerated doses, and both trials were discontinued. Reverse translational studies in mice demonstrated that IACS-010759 induced behavioral and physiological changes indicative of peripheral neuropathy, which were minimized with the co-administration of a histone deacetylase 6 inhibitor. Additional studies are needed to elucidate the association between OXPHOS inhibition and neurotoxicity, and caution is warranted in the continued development of complex I inhibitors as antitumor agents.

INTRODUCTION

Targeting oxidative phosphorylation (OXPHOS)^1–6 has emerged as an attractive anti-cancer strategy due to the dependency of certain tumors on this metabolic pathway^5,7–9. This approach, however, has been limited by poor potency (for example biguanides^10–12), off-target pharmacology (for example rotenone^13,14), a suboptimal pharmacokinetic (PK) profile¹⁵, and dose-limiting toxicities (for example oligomycin, rotenone¹⁴, BAY 87–2243⁵, ASP4132¹⁶). Previously, we reported the discovery of IACS-010759, a highly potent and selective small-molecule complex I inhibitor with favorable attributes for clinical evaluation¹⁷. Preclinical studies confirmed that IACS-010759 selectively inhibited mitochondrial complex I by binding the ND1 subunit at the entrance to the quinone binding channel^17,18. Efficacious antitumor doses of IACS-010759 in multiple preclinical models of OXPHOS-dependent cancers were well tolerated, with no observed neurotoxicity, and induced energetic stress and decreased aspartate production¹⁷. This resulted in reduced cell viability and apoptosis, and ultimately extended mouse survival¹⁷. Additionally, IACS-010759 administration increased plasma lactate in responsive tumor cells¹⁷, indicating a compensatory increase in glycolysis and, consequently, therapeutic opportunities for targeting OXPHOS in glycolysis-deficient tumors.

Based on these preclinical findings¹⁷, IACS-010759 was assessed in two Phase I trials. The first-in-human study evaluated patients with relapsed/refractory acute myeloid leukemia (AML), a malignancy strongly dependent on OXPHOS^19–21. The second trial, which leveraged pharmacokinetic data from the AML study to inform on dosing regimens, enrolled patients with advanced solid tumors, with an emphasis on glycolysis-deficient cancers such as those with ENO1 loss¹⁷ and SMARCA1 mutations²². Trial results revealed that target IACS-010759 plasma exposures were challenging to maintain due to intermittent dosing schedules and drug interruptions, the latter necessary to mitigate adverse events related to elevated blood lactate and neurotoxicity. Consequently, IACS-010759 achieved a modest, transient suppression of OXPHOS and limited antitumor activity, and both trials were discontinued. Follow-up studies confirmed that clinically achievable exposures were too low to induce therapeutic benefit, and escalation to efficacious doses in mice resulted in peripheral neuropathy. Coadministration with an HDAC6 inhibitor mitigated pain associated with IACS-010759-induced peripheral neuropathy in mouse models. Overall, our findings reveal significant risks associated with the clinical development of complex I inhibitors.

RESULTS

Study design and objectives

Two independent, open-label, dose escalation Phase I trials (Fig. 1) were conducted at The University of Texas MD Anderson Cancer Center in 17 adult patients with relapsed/refractory AML (2016; NCT02882321; ‘AML trial’) and in 23 adult patients with advanced solid tumors (2017; NCT03291938; ‘Solid Tumor trial’). For both trials, the primary objectives were to determine the safety, tolerability, maximum tolerated dose, and recommended phase 2 dose of IACS-010759; secondary objectives were to evaluate the pharmacokinetics, pharmacodynamics and preliminary antitumor activity of IACS-010759. Both preclinical and AML trial data were used to inform the Solid Tumor trial dosing schedules. Dosing regimens are provided in Methods and Supplementary Table 1; patient characteristics are presented in Table 1 and Supplementary Tables 2–6.

Figure 1. Flow diagram summarizing the AML and Solid Tumor trials and clinical analyses.

Flow diagram summarizing the enrollment and dose scheduling of the AML (left) and Solid Tumor (right) trials as well as the pharmacokinetic and pharmacodynamic analyses performed on blasts from AML patients. QD, once daily. OCR, oxygen consumption rate.

Table 1.

Patient demographics and characteristics; data presented as n (%) [95% confidence interval].

Factor	Category	AML (n = 17)	Solid Tumors (n = 23)
Median age, years (range)		60 (29–77)	53 (23–71)
Sex	Male	10 (59) [33, 82]	20 (87) [66, 97]
	Female	7 (41) [18, 67]	3 (13) [3, 34]
ECOG Performance Status	0	3 (18) [3.8, 43]	2 (9) [1, 28]
	1	10 (59) [33, 82]	21 (91) [72, 99]
	2	4 (24) [7, 50]	0
European Leukemia Net for AML	Intermediate	4 (24) [7, 50]	-
	Adverse risk	13 (76) [50, 93]	-
Tumor	Adenoid Cystic Carcinoma	-	1 (4) [0.1, 22]
	Castration-resistant prostate cancer	-	4 (17) [5, 39]
	Cholangiocarcinoma	-	2 (9) [1, 28]
	Chondrosarcoma	-	1 (4) [0.1, 22]
	Colorectal	-	4 (17) [5, 39]
	Glioblastoma	-	4 (17) [5, 39]
	Malignant granular cell tumor of the neck	-	1 (4) [0.1, 22]
	Maxillary sinus squamous cell carcinoma	-	1 (4) [0.1, 22]
	Non-Small Cell Carcinoma	-	1 (4) [0.1, 22]
	Pancreatic adenocarcinoma	-	3 (13) [3, 34]
	Spindle cell sarcoma	-	1 (4) [0.1, 22]
Prior therapies	Median (range)	4 (1–10)	3 (1–9)
	Median targeted therapy (range)		2 (1–6)
	Median immunotherapy (range)		1 (1–3)
	Median chemotherapy (range)	2	3 (1–6)
	Hypomethylator (HMA)	2	-
	HMA and chemotherapy	12	-
	Investigational therapy	8	-
	Stem-cell transplantation	5	-
Cytogenetic category	Normal (Diploid)	5 (29) [10, 56]	-
	Complex	11 (65) [38, 86]	-
	Chromosome 7 aberrations	1 (6) [0.2, 29]	-
WBC count, x109/L (range)		3.7 (0.1–13.9)	-
Peripheral blood blasts (range)		39 (0–94)	-
Bone marrow blasts (range)		54 (20–95)	-
De Novo AML		13	-
Secondary AML		4	-

Data presented as n (%) [95% confidence intervals] unless otherwise indicated.

Drug-related toxicities in patients with AML and solid tumors

Adverse events were monitored at scheduled timepoints throughout each trial. Treatment-emergent adverse effects (TEAEs) and treatment-related adverse events (TRAEs) were reported in both trials (Supplementary Tables 7–10). Toxicities related to elevated blood lactate or neurotoxicity emerged as the most common adverse events and contributed to the premature termination of both trials. Specifically, common grade 1–2 TRAEs included elevated blood lactate (35% AML; 83% Solid Tumor), lactic acidosis (29% AML), nausea (29% AML, 65% Solid Tumor), vomiting (18% AML, 30% Solid Tumor), myalgia (6% AML, 30% Solid Tumor), and peripheral neuropathy (12% AML, 35% Solid Tumor). Grade ≥3 TRAEs in both trials included elevated blood lactate (53% AML, 9% Solid Tumor), lactic acidosis (24% AML), nausea (9% Solid Tumor), vomiting (13% Solid Tumor), and peripheral neuropathy (6% AML, 4% Solid Tumor). One Solid Tumor trial patient developed grade 3 visual impairment. All adverse events leading to treatment discontinuation are detailed in Supplementary Tables 11–14.

Elevation of blood lactate has frequently been reported following OXPHOS inhibition^{2,16,17,23,24}. Here, eight out of 16 AML trial patients who developed increased blood lactate also developed lactic acidosis (47.1%), with 7/8 cases determined as treatment-related and 4/8 cases reported as grade ≥3 (Supplementary Table 7, 9). Further, 20 Solid Tumor trial patients (87%) had treatment-related increases in blood lactate without lactic acidosis (Supplementary Table 10). Increasing IACS-010759 exposure was associated with increasing plasma lactate, but not with decreasing blood pH (Extended Data Fig. 1), and drug exposures of >8 nM (4.5 ng/mL equivalent) were associated with increased probability of elevated lactate (Fig. 2a,b). Elevated blood lactate did not result in any fatal events but did lead to treatment discontinuation for one Solid Tumor trial patient (Supplementary Table 14).

Figure 2. Drug-related toxicity.

a-b, Change in venous lactate levels across plasma IACS-010759 concentrations in AML (a) and Solid Tumor (b) cohorts. Each dot represents the mean value of all samples collected from the cohort at the same time point. c, Study timelines of patients from the Solid Tumor (red) and AML (blue) trials. Diamonds, onset of peripheral neuropathy; circles, onset of myalgia. Patients with SMARCA1-null or ENO1-null tumors are noted. Dosing regimen indicated on the right. d, (left) Plasma lactate levels in Solid Tumor patients who developed (red) or did not develop (blue) peripheral neuropathy. Data analyzed by two-way ANOVA, *P = 0.0040. Data show mean ± SE; n=11 biologically independent samples. Individual comparisons of plasma lactate levels in Solid Tumor patients who (center) developed or (right) did not develop peripheral neuropathy. e, (left) Plasma IACS-010759 concentrations in Solid Tumor patients who developed (red; n=11 biologically independent samples) or did not develop (blue; n=9 biologically independent samples) peripheral neuropathy. Data analyzed by two-way ANOVA, showing mean ± SE. Individual comparisons of plasma IACS-010759 levels in Solid Tumor patients who (center) developed or (right) did not develop peripheral neuropathy.

Neurotoxicity emerged as a prominent TRAE in both trials, with grade 1–3 peripheral neuropathy observed in 11 Solid Tumor and four AML trial patients (Table 2 and Supplementary Tables 9 and 10). In Solid Tumor trial patients, symptoms of peripheral neuropathy included grade 1–3 hand and/or feet and/or leg/hip numbness, with 3/11 patients developing grade 1–3 weakness and 3/11 patients developing grade 1–2 paresthesia. Painful neuropathy was not observed. Interestingly, 5/11 Solid Tumor patients who developed peripheral neuropathy also reported myalgia, onset of which preceded peripheral neuropathy in all cases (Fig. 2c). In contrast, onset of myalgia was not observed in any of the four AML trial patients who developed peripheral neuropathy (Fig. 2c). Nerve biopsies from two Solid Tumor trial patients with grade 3 neuropathy revealed mixed axonal and demyelinating neuropathy, with involvement of large and small fibers (axons) in one patient as well as histology consistent with axonal neuropathy and loss of large myelinated fibers in the other (Extended Data Fig. 2). Overall, the incidence of peripheral neuropathy increased with dosage, dose frequency, and duration on trial (Table 2, Fig. 2c), thus preventing further dose and schedule escalation. Recovery from neuropathy varied among patients, but generally occurred gradually after drug discontinuation. Interestingly, elevated plasma lactate (p=0.004), but not IACS-010759 exposure, was associated with peripheral neuropathy in Solid Tumor trial patients (Fig. 2d,e). Previous allogeneic stem cell transplantation (allo-SCT) significantly (p=0.002) increased the risk of developing peripheral neuropathy in AML trial patients, while prior cytotoxic chemotherapy (p=0.005) and/or concomitant diagnosis of diabetes mellitus (p=0.043) increased the risk of peripheral neuropathy in Solid Tumor trial patients (Table 2). Additionally, one Solid Tumor trial patient developed grade 3 visual impairment, possibly suggesting optic nerve toxicity.

Table 2. Clinical characteristics and the onset of neurotoxicity in the 40 patients who received IACS-010759 treatment.

		Solid Tumors				AML
		NP (N=11)	Non-NP (N=12)	Chi-square statistic	p-value	NP (N=6)	Non-NP (N=11)	Chi-square statistic	p-value
Factor	Category
Risk Factors for NP (N, %, 95% confidence interval)	Prior chemotherapy: platinum or taxanes	11 (100) [72, 100]	4 (33) [10–65]	7.988	0.005	1 (17) [0.4–64]	-	-	-
	Prior stem cell transplantation	-	-	-	-	5 (83) [36, 100]	1 (9) [0.2, 41]	9.370	0.002
	Diabetes mellitus	5 (45) [17, 77]	1 (8) [0.2, 38]	4.102	0.043	1 (17) [0.4, 64]	5 (45) [17, 77]	1.410	0.235
	Hypothyroidism	1 (9) [0.2, 41]	1 (8) [0.2, 38]	0.004	0.949	-	2 (18) [2, 52]	-	-
	Prior checkpoint inhibitor	4 (36) [11, 69]	2 (17) [2, 48]	1.154	0.283	1 (17) [0.4, 64]	1 (9) [0.2, 41]	0.215	0.643
Lactate (mmol/L)	Average lactate level before OXPHOS	1.2	1.4	-	-	1.3	1.3	-	-
	Average highest lactate level during OXPHOS	4.5	2.6	-	-	3.9	3.0	-	-
Glycemia (mg/dL)	Level before OXPHOS	144	92			114	119
	Level>140 (n)	5	0	4.102	0.428	1	3	0.243	0.622
	Level<140 (n)	6	12			5	8
IACS dosing (N, %, 95% confidence interval)	0.5 mg/day	-	-			-	5 (45) [17, 77]
	1.0 mg/day	-	2 (17) [2, 48]			2 (33) [4, 78]	1 (9) [0.2, 41]
	1.5 mg/day	-	2 (17) [2, 48]			-	-	0.701	0.402
	2.0 mg/day	4 (36) [11, 69]	7 (58) [28, 85]	1.983	0.159	1 (17) [0.4, 64]	2 (18) [2, 52]
	2.5 mg/day	4 (36) [11,69]	1 (8) [0.2, 38]			3 (50) [12, 88]	3 (27) [6, 61]
	3.0 mg/day	3 (27) [6, 61]	-			-	-
Neuropathy (N, %, 95% confidence interval)	Overall	11 (48) [27, 69]	12 (52) [31, 73]			6 (35) [14, 62]	11 (65) [38, 86]

Abbreviations: N, number of patients; NP, neuropathy; %, percentage; [95% confidence intervals]. Data analyzed by Chi-square test; effect sizes:

Efficacy

Responses in AML and Solid Tumor trial patients were assessed according to the guidelines of European LeukemiaNet 201725 and RECISTv.1.126, respectively. No patient in the AML trial responded to IACS-01059 treatment. One patient in Solid Tumor cohort 4 with advanced castration-resistant prostate cancer achieved a confirmed RECISTv1.1 partial response with resolution of cancer-related pain, and remained on study for four months (Supplementary Fig. 1). An additional eight patients in the Solid Tumor trial patients had a best response of RECISTv1.1 stable disease (Supplementary Table 15). No RECISTv1.1 stable disease or objective responses were achieved in any of the three patients with ENO1 loss, nor in the two patients with SMARCA1 deletions. The observed modest efficacy, coupled with adverse event profiles, supported the discontinuation of both trials (Supplementary Table 15,16).

Pharmacokinetics of IACS-010759

Consistent with preclinical data¹⁷, plasma levels of IACS-010759 in patients increased in a dose-dependent manner and demonstrated a long terminal half-life of approximately 16 hours (Extended Data Fig. 3; Supplementary Table 17). Data from preclinical¹⁷ and investigational new-drug studies using intermittent dosing schedules indicated that plasma exposures needed to reach a minimum of ~20 nM between doses (C_min) to achieve antitumor activity in patients. Repeated dosing over the 21-day daily dosing cycle enabled gradual accumulation of IACS-010759 concentrations to accumulate slowly in the first two AML cohorts and approach target efficacious concentration in AML Cohort 2 by the end of the first cycle, but a steady state was not achieved (Extended Data Fig 3a; Extended Data Fig. 4a).

Due to the long half-life and slow accumulation of IACS-010759 as well as the frequency of drug-related toxicities in Cohorts 1 and 2, an induction-maintenance dosing strategy was implemented in AML Cohorts 3 and 4 as well as all Solid Tumor cohorts (Fig. 1, Supplementary Table 1). This change enabled IACS-010759 exposures to approach target levels during the induction phase without accumulation during the maintenance phase in most patients in AML cohorts 3 and 4, as well as in all patients in the Solid Tumor trial (Extended Data Fig. 3b–d, Extended Data Fig. 4b–f). In general, dosing two or three times per week maintained IACS-010759 plasma levels better than once-weekly dosing (Extended Data Fig. 3b–d, Extended Data Fig. 4b–f). Regardless, IACS-010759 levels of ≥20 nM were not maintained with any dosing schedule, and efforts to increase drug exposure were obstructed by dose holds necessary for managing drug-related toxicities. Additional PK properties are presented in Supplementary Table 17.

Evidence of target inhibition in AML blasts

We evaluated the pharmacodynamic markers of target inhibition and the biological response of tumor cells to IACS-010759 by isolating blasts from patients with AML. Our previous findings demonstrated sensitivity to IACS-010759 in primary blasts as well as in patient-derived xenograft AML models and multiple AML cell lines¹⁷. Here, baseline (p=0.001) and maximal (p=0.003) oxygen consumption rates (OCR) were consistently and significantly lower than pre-treatment OCR at the end of the first 7-day QD dosing, with a >25% reduction in baseline and maximal OCR observed in blasts from 6/12 and 8/12 patients, respectively (Fig. 3a,b). Among these patients with AML, decreases in baseline and maximal OCR were not maintained in 2/6 patients and in 3/8 patients, respectively (Fig. 4a,b), which aligned with reduced exposures during maintenance in AML Cohorts 3 and 4 (Extended Data Fig. 3b; Extended Data Fig. 4b). Individually, correlations between IACS-010759 exposure and OCR varied (Extended Data Fig. 5, 6), with a significant correlation between plasma concentration and baseline or maximal OCR in three patients (Extended Data Fig. 5a,c,k) or five patients (Extended Data Fig. 6a,e,h,k,l), respectively, as well as between plasma concentration and both baseline and maximal OCR in two patients (Extended Data Fig. 5a,k; Extended Data Fig. 6a,k). Poor correlation between plasma concentration and OCR was observed in four patients (Extended Data Fig. 5b,c,f,g, Extended Data Fig. 6bc,f,g), suggesting onset of resistance to IACS-010759, such as an increase in mitochondria within the blast to compensate for complex I inhibition²⁷. Indeed, a significant increase in the ratio between mitochondrial and genomic DNA (p=0.04) was identified in blasts from most patients with AML by longitudinal Q-PCR analysis (Fig. 3c), strongly suggesting rapid adaptation to IACS-010759 treatment.

Figure 3. Evidence of target inhibition in AML blasts.

a-b, Change in (a) baseline and (b) maximal oxygen consumption rate (OCR) prior to dosing (C1D1), after one week of QD dosing (C1D7/C1D14), or extended dosing (C1D15/21/28). n=12 biologically independent samples. Data analyzed by a two-sided paired Student’s t-test. c, The ratio of mitochondrial (mtDNA) to genomic DNA (gDNA) from AML blasts collected pre- and post-dose on Day 16/21/27/28. Data analyzed with a two-sided paired Student’s T-test; mean ± SE shown. Paired patient samples are indicated; n=8 biologically independent samples. d, Baseline OCR across IACS-010759 concentrations in AML blasts from Patients 16, 17, and 19. Grey line indicates expected modulation of baseline OCR by IACS-010759¹⁷. All samples are biologically independent. Patients 16 and 17: n=6. Patient 19: n=3 at C1D1, C1D1(4); n=4 at C1D7, C1D7(4); n=6 at all other times. Mean ± SE shown. e, Schematic depicting effects of IACS-010759 on oxidative metabolism and alternative fuel pathways. TCA = The tricarboxylic acid cycle; GSH = glutathione; ETC = electron transport chain. f-h, IACS-010759 exposure on (f) NMP, (g) NDP, and (h) NTP in AML blasts from Patient 17. n=7, n=6, or n=7 biologically independent samples for (f), (g), and (h), respectively. Data analyzed by a simple linear regression. i, IACS-010759 exposure on aspartate in AML blasts from patients 16, 17, and 19. N=4, n=7, or n=5 biologically independent samples for Patients 16, 17, or 19 respectively. Data analyzed by a simple linear regression. j, IACS-010759 exposure on lactate in AML blasts from patients 16, 17, and 19. N=4, n=7, or n=5 biologically independent samples for Patients 16, 17, or 19 respectively. Data analyzed by a simple linear regression. k, (top) The OXPHOS signature was generated from published preclinical data¹⁷ and applied to AML blasts from Patients 16, 17, and 19. (bottom) The 1065 signature was established using mRNAs significantly modulated in AML blasts from Patients 16, 17, and 19 upon IACS-010759 treatment.

Figure 4. IACS-010759 induces physiological and behavioral symptoms of peripheral neuropathy in preclinical models.

a, Plasma IACS-010759 concentrations in NSG mice treated with escalating doses of IACS-010759. Mean ± SE shown. Data analyzed with a 2-way analysis of variance (ANOVA) with a Tukey’s multiple comparison test; n=3 biologically independent samples. At 2 hr: ***p<0.0001 for 5 vs 0.1, 0.3, or 1 mg/kg; *p=0.0129 for 1 vs 0.1 mg/kg; **p=0.0059 for 1 vs 0.3 mg/kg. At 4 hr: **p<0.0001 for 5 vs 0.1, 0.3, or 1 mg/kg; *p=0.0004 for 1 vs 0.1 mg/kg; ***p=0.0003 for 1 vs 0.03 mg/kg. At 8 hr: ***p<0.0001 for 5 vs 0.1, 0.3, or 1 mg/kg; *p=0.340 for 1 vs 0.3 mg/kg. n.s. = non-significant. b, Baseline oxygen consumption rates (OCR) from blasts from the spleen of NSG mice treated with vehicle or escalating doses of IACS-010759. N=5 biologically independent samples. Data analyzed with a two-sided unpaired Student’s t-tests; ns = non-significant. c, Kaplan-Meier survival analysis of NSG mice treated with escalating doses of IACS-010759 or vehicle. N=9 mice/group. Data analyzed with a Log-rank test; vehicle vs 0.03 mg/kg: **p=0.0059; vs 1 mg/kg *p=0.0070; vs 5 mg/kg ***p=0.0004. d, Mechanical sensitivity of hind paws of mice (n=4/group) treated with IACS-010759 or vehicle for 5 days on (grey), 2 days off. Differences from controls (vehicle) were analyzed with a two-way ANOVA with a Dunnett’s multiple comparison test. Mean ± SE shown. e, OCR of murine dorsal root ganglion (DRG) after the last dose of IACS-010759 or vehicle +/− ACY-1215. n=8 biologically independent samples. Data analyzed by two-way ANOVA with Sidak’s multiple comparison test; n.s. = non-significant. Mean ± SE shown. f, Mean maximal OCR of murine DRG neurons after the last dose of IACS-010759 or vehicle. n=8 or n=4 biologically independent samples for 0 and 1 mg/kg or 0.3 mg/kg IACS-010759, respectively. Data analyzed by one-way ANOVA with Dunnett’s multiple comparison test. Mean ± SE shown. g, Mechanical sensitivity of murine hind paws (n=4/group) treated with IACS-010759 (0.3 mg/kg or 1 mg/kg) or vehicle +/− ACY-1215 for 5 days on (grey) and 2 days off. Mean ± SE shown. Data analyzed by two-way ANOVA with Sidak’s multiple comparison test.

Next, the relationship between exposure and the biological response of blasts to IACS-010759 was determined. For this, we compared response to IACS-010759 in blasts collected from AML Patients 17, 19, and 16, who received the same dosing regimen (Supplemental Table 1) yet achieved differential exposure and demonstrated relatively strong, mild, or weak drug-induced reductions in baseline OCR, respectively (Fig. 3d). We previously showed that IACS-010759 altered levels of metabolites related to complex I inhibition of and reduced energetic status¹⁷ (Fig. 3e), including 1) inhibiting the production of nucleoside triphosphates (NTPs); 2) inhibiting oxidation of NADH to NAD+; 3) elevation of glutamine, which can be shuttled into alternative fuel pathways in lieu of OXPHOS and aspartate synthesis; and 4) elevating levels of glycolytic endpoints. Here, blasts from Patient 17, who achieved the relatively highest exposure, demonstrated metabolite changes indicating complex I inhibition and reduced energetic status. Specifically, upon IACS-010759 treatment, intracellular levels of nucleoside monophosphates (NMPs; p<0.01) and diphosphates (NDPs; p<0.01) significantly increased while NTPs levels significantly decreased (p<0.01) (Fig. 3f–h), suggesting a decrease in NTP production. Further, NAD+ levels significantly decreased (p = 0.03) while those of tryptophan (p = 0.08) and nicotinamide (p = 0.002), metabolic precursors of NAD+, significantly increased (Extended Data Fig. 7a–c), suggesting decreased NADH oxidation. Glutamine levels trended upward (p = 0.08) (Extended Data Fig. 7d) while aspartate levels trended downward (p=0.06) (Fig. 3i), consistent with increased investment in alternative fuel pathways and decreased investment in cell growth. Finally, alanine levels significantly increased (p = 0.046; Extended Data Fig. 7e) while lactate levels did not change (Fig. 3j), suggesting a general elevation in glycolysis upon complex I inhibition. In contrast, blasts from Patient 19, who experienced lower IACS-010759 exposure than Patient 17, demonstrated more moderate metabolite changes that were consistent with complex I inhibition and reduced energetic status. Specifically, IACS-010759 significantly reduced NTP levels (p<0.05) but did not affect NMP and NDP levels in a manner consistent with complex I inhibition (Extended Data Fig. 7f). Although nicotinamide levels significantly increased (p=0.006; Extended Data Fig. 7b) and tryptophan trended upward (Extended Data Fig. 7c), NAD+ levels significantly increased (p=0003; Extended Data Fig. 7a), suggesting potential complex I activity. Further, significantly decreased glutamine (p=0.02; Extended Data Fig. 7d), unaffected aspartate (Fig. 3i), unaffected alanine (Extended Data Fig. 7e), and significantly increased lactate (p= 0.005; Fig. 3j) levels upon treatment suggested no increased activation of alternative fuel pathways. Finally, blasts from Patient 16, who achieved the relatively weakest IACS-010759 exposure, did not demonstrate metabolite changes consistent with impaired complex I activity or reduced energetic status. Specifically, IACS-010759 treatment did not affect levels of NMP, NDP, and NTP (Extended Data Fig. 7g) nor levels of NAD+ (Extended Data Fig. 7a), nicotinamide (Extended Data Fig. Fig 7b), and tryptophan (Extended Data Fig. Fig 7c). Levels of glutamine (Extended Data Fig. Fig 7d), lactate (Fig. 3j), and alanine (Extended Data Fig. 7e) were unaffected, and levels of aspartate were significantly elevated (p=0.02; Fig. 3i) upon IACS-010759 treatment, suggesting no increase in the activation of alternative fuel pathways. Overall, these metabolite data suggest that high exposure of IACS-010759 is needed to modulate blast biology in a manner that indicates complex I inhibition and low energetic status.

To evaluate gene modulation by IACS-010759 in blasts from Patients 16, 17, and 19, we first conducted hierarchical clustering using our published OXPHOS signature generated from preclinical data¹⁷ and a new signature with 1065 mRNAs that were significantly modulated in blasts upon IACS-010759 treatment (“1065 signature”) (Fig. 3k). Among the 176 genes identified in the OXPHOS signature, 166 genes were present in our current patient RNA sequencing (RNA-seq) dataset, and 11 genes overlapped with the genes in the 1065 signature. Interestingly, unlike treatment-induced modulation of OCR and metabolites (Fig. 3d, f–j; Extended Data Fig. 7), modulation of gene expression across both OXPHOS and 1065 signatures was consistent in all three AML patients. Next, we conducted Gene Ontology (GO) enrichment analysis on RNA-seq results from blasts collected from Patients 16, 17, and 19 at various timepoints pre- and post-dose across Cycle 1. In all post-dose samples, mRNAs related to chromatin organization and DNA damage response were downregulated, and terms related to RNA/DNA metabolism and transport were enriched (Extended Data Fig. 8a). Pathway analysis confirmed upregulation of pathways related to transfer RNA biosynthesis and downregulation of pathways related to cell growth, RTK/PI3K signaling, and proliferation upon IACS-010759 treatment in blasts from all three patients, regardless of exposure (Extended Data Fig. 8b). Overall, IACS-010759-induced changes in gene expression align with preclinical findings that show increased DNA damage as well as inhibited nucleotide biosynthesis and cell proliferation upon treatment¹⁷, but do not align with treatment-induced changes in exposure and metabolites.

Our findings suggest that low exposures of IACS-010759 can induce transcriptomic changes associated with OXPHOS inhibition in AML blasts, but high exposures are needed to modulate biological activity in a manner consistent with complex I inhibition. Unfortunately, treatment-induced changes in blasts were insufficient to reduce AML disease burden.

Reverse Translational Studies

As previously described, the target exposure of 20 nM was not maintained in either trial (Extended Data Fig. 3, 4), and observed clinical exposures induced limited antitumor activity and frequent adverse events related to elevated lactate and neurotoxicity (Supplementary Fig. 1 Supplementary Tables 7–10). Reverse translational studies were thus conducted to investigate the efficacy and toxicities induced by doses aligned with observed clinical exposures of IACS-010759 as well as to evaluate strategies for mitigating treatment-induced toxicities.

A pharmacokinetics study assessing escalating doses of IACS-010759 administered on a 5-days on/2-days off schedule in NSG and C56Bl6 mice was first performed. Doses of 0.1 and 0.3 mg/kg generated Cmax exposures similar to the highest exposures achieved in AML and Solid Tumor clinical trials (Fig. 4a), and, consistent with blast data, induced moderate modulation of baseline OCR (Fig. 4b). However, only doses of 1 or 5 mg/kg extended median survival in mice by 7 and 35 days, respectively (Fig. 4c). Plasma lactate was elevated by all doses in C56Bl6 mice, but only elevated by the 5 mg/kg dose in NSG mice (Extended Data Fig. 9a). Consistent with clinical data, these findings confirm that clinically achievable IACS-010759 exposures modestly modulate OCR, elevate lactate, and are not efficacious.

To investigate the relationship between IACS-010759 and behavioral symptoms of neurotoxicity, C57/Bl6 mice were treated with escalating doses of IACS-010759 on a 5-days on/2-days off schedule for two weeks. IACS-010759 induced mechanical allodynia, a behavioral symptom of neuropathy that can be measured using von Frey hairs²⁸, in a dose-dependent manner that did not resolve by two weeks after the last dose (Fig. 4d). Further, IACS-010759-treated mice experienced spontaneous pain, as a conditioned place preference (CPP) paradigm found that IACS-010759-treated mice spent significantly more time in the chamber associated with pain relief than vehicle-treated mice (Extended Data Fig. 9b,c). Additionally, a significant decrease in balance and fine motor function, but no change on motivation to perform the task, was observed in IACS-010759-treated mice compared to vehicle-treated mice, as measured by a beam walk test²⁹ (Extended Data Fig. 9d). Overall these findings demonstrate that, similar to clinical findings, IACS-010759 induces signs of peripheral neuropathy at a clinically relevant exposure.

Next, we investigated the association between peripheral neuropathy and mitochondrial dysfunction in peripheral sensory neurons. Strikingly, IACS-010759 treatment dose-dependently inhibited OCR in dorsal root ganglia (DRG) collected 3–4 hours after the last dose (Fig 4e) and at 5 weeks after the last dose (Fig 4f, Extended Data Fig. 9e). The highest dose of IACS-010759 also reduced intraepidermal nerve fiber density (IENF) in the hind paw skin, a marker of peripheral neuropathy that is sensitive to mitochondrial dysfunction^30,31 (Extended Data Fig. 9f). Expression of ATF3, a transcription factor induced after traumatic nerve injury³², did not increase following IACS-010759 treatment, indicating that neuronal degeneration had not occurred (Extended Data Fig. 9g). Finally, electronic microscopy analysis of the murine sciatic nerve at five weeks after completion of vehicle or 5 mg/kg IACS-010759 found that IACS-010759 resulted in myelin damage characterized by the decompaction of myelin and split sheets (Extended Data Fig. 9h), which was consistent with clinical data.

In an attempt to assess mitigation strategies for the peripheral neuropathy induced by pharmacological complex I inhibition, we coadministered IACS-010759 with the experimental HDAC6 inhibitor ACY-1215 (Ricolinostat), which has been shown to prevent and reverse chemotherapy-induced peripheral neuropathy33,34. C57/Bl6 mice were treated with 0.3 or 1 mg/kg of IACS-010759 with or without 30 mg/kg of ACY-1215, or with vehicle, for two weeks on a 5-days-on/2-days-off schedule. Behavioral and physiological markers of peripheral neuropathy were assessed 4 days after the last dosing. Co-administration of ACY-1215 significantly attenuated IACS-010759-induced mechanical allodynia (Fig. 4g), and prevented IACS-010759-induced spontaneous pain (Extended Data Fig. 10a), sensorimotor deficits (Extended Data Fig. 10b), and damage to the myelin of the sciatic nerve (Extended Data Fig. 10c). However, co-administration with ACY-1215 did not prevent IACS-010759-induced reduction of OCR in the DRG (Fig. 4e). Together, these data suggest that IACS-010759-induced peripheral neuropathy may be mediated by multiple mechanisms but some symptoms associated with this neurotoxicity can be prevented by coadministration of ACY-1215.

Overall, our reverse translational studies confirm that the anti-tumor efficacy of IACS-010759 was limited by the development of peripheral neuropathy as well as provide guidance for assessing the neurotoxicity of OXPHOS inhibitors at the preclinical stage. Additional studies are needed to investigate the underlying mechanisms linking OXPHOS inhibition with the peripheral neuropathy phenotype.

DISCUSSION

Based on preclinical findings¹⁷, first-in-human Phase I clinical trials were initiated to assess the safety and efficacy of IACS-010759, a selective, small-molecule complex I inhibitor, in patients with AML or advanced solid tumors. Clinical findings revealed that implemented dosing regimens could not maintain efficacious levels of plasma IACS-010759, resulting in limited antitumor activity despite achieving on-target inhibition. Further, IACS-010759 induced resistance mechanisms in patients with AML, with evidence suggesting a compensatory increase in mitochondria in blasts. This may have been mediated by mitochondrial fission and/or acquisition from bone marrow mesenchymal stem cells via tunneling nanotubes²⁷. Overall, our data indicated that patients needed to achieve and maintain exposures higher than those observed during the trials to clinically benefit from IACS-010759.

Mechanism-based toxicities related to elevated blood lactate and neurotoxicity, including lactic acidosis, nausea, vomiting, and peripheral neuropathy, prevented further dose escalation and schedule intensification of IACS-010759. These toxicities have also limited the translation of other complex I inhibitors, such as ASP4132¹⁶ and BAY 87–2243^5,35, for cancer treatment. Additionally, widespread low-grade adverse events that included nausea and vomiting contributed to preventing dose escalation in phase I trials of IM156, a complex I-targeting biguanide that demonstrated moderate efficacy against refractory solid tumors¹². Here, persistent and dose-dependent IACS-010759-induced peripheral neuropathy was the most serious dose-limiting toxicity and contributor to the early termination of both trials. Follow-up studies confirmed that IACS-010759 doses high enough to enhance median survival in mice also elevated blood lactate as well as induced behaviors indicative of peripheral neuropathy and damaged the myelin of peripheral nerves. Our findings suggest that all investigational agents targeting complex I and similar mechanisms should be systematically assessed for neurotoxicity at the preclinical stage.

The association between OXPHOS inhibition and disorders affecting the nervous system has been observed in individuals treated with other mitotoxic agents^30,36. For instance, several studies support mitochondrial dysfunction in primary sensory neurons as a main contributor to chemotherapy-induced peripheral neuropathy^{30,37,38,39,40,41}. Chronic inhibition of complex I by the pesticide rotenone has been shown to induce symptoms associated with Parkinson’s Disease in rodent models¹⁴. Further, elevated blood lactate and disorders^42–45 related to energetically expensive tissues, including neural^43,46, muscle^43,46, and cardiac^47–49, are common in individuals with congenital complex I dysregulation, although were not observed in our patients. While it is difficult to compare congenital with pharmacologically induced effects, this discrepancy may suggest that IACS-010759 treatment durations were too short for these toxicities to emerge, as well as potentially reflect the limited number of enrolled patients and/or differences in the rate of tissue uptake and drug penetration between an oral drug and congenital condition. Regardless, the future development of anti-cancer drugs targeting the OXPHOS pathway should monitor for toxicity on tissues highly dependent on aerobic respiration.

Elevated lactate is commonly observed upon OXPHOS inhibition^{2,16,17,23,24} and indicates a compensatory increase in glycolysis to mitigate energetic stress^36,50. In our patients, elevated lactate was a stronger predictor of peripheral neuropathy than IACS-010759 exposure. Elevated lactate due to a metabolic shift towards glycolysis has been linked to increased inflammation-driven pain, including that observed during peripheral neuropathy⁵¹. Interestingly, our reverse translational studies demonstrated that coadministration of an HDAC6 inhibitor-which can reverse murine chemotherapy-induced mechanical hypersensitivity by promoting anti-inflammatory cytokine signaling in sensory neurons and improving axonal mitochondrial health33,34–prevented IACS-010759-induced behavioral changes indicative of peripheral neuropathy. While additional studies are needed to elucidate the association between elevated lactate and inflammation-driven pain in sensory neurons, our data, in addition to other studies^{33,34,37,39,52}, suggest that combination approaches may help mitigate the neurotoxic effects associated with OXPHOS inhibitors.

Interestingly, the predisposing risk factors for developing IACS-010759-induced peripheral neuropathy were also associated with neurotoxicity, suggesting sensitization to, or existing, neural damage in some patients before enrollment. Among patients with AML, alloSCT was the strongest predictor for developing peripheral neuropathy. While rare, alloSCT has been associated with neurological complications in patients with hematologic malignacies^53–56. This association is largely attributed to chronic graft-versus-host disease^53–55, but contributing factors include immune system reconstitution⁵³, drug complications^53,56, prior neurological disease^53,56, the female sex⁵⁷, and total body irradiation⁵⁷. Among patients in the Solid Tumor trial, prior chemotherapy and concomitant diagnosis of diabetes mellitus, both widely associated with peripheral neuropathy^30,40, were the strongest predictors for developing peripheral neuropathy. Together, our findings suggest that future trials assessing complex I-targeting agents in patients with cancer should be aware of previous therapies or pre-existing conditions that may have caused neural damage.

Overall, our findings raise concerns about the risks and feasibility of leveraging complex I inhibitors for anti-cancer therapy, and suggest that more research is needed to elucidate the effects of these drugs on normal tissues heavily dependent on the OXPHOS pathway. As complex I inhibitors continue to be evaluated as anti-cancer agents^5,47, we urge researchers to critically and comprehensively assess for the toxicity of their mitotoxic agents prior to advancing the translation of their compound.

METHODS

Patient selection, screening, and eligibility

Patients eligible for the AML trial were previously diagnosed with AML using the World Health Organization criteria, in addition to having either failed previous induction therapy (refractory AML), had a first relapse after previous therapy with a first remission duration of <12 months or had relapsed to more than one previous AML therapy. Additional eligibility criteria included 1) age 18+ years; 2) Eastern Cooperative Oncology Group performance status ≤2; 3) completion of prior cytotoxic/non-cytotoxic agents and biological/immune-based therapies, including investigational agents, occurred at least 2 weeks or 5 half-lives interval –whichever was shorter– prior to start of trial; 4) adequate organ function (total bilirubin ≤2.0 x the upper limit of normal (ULN) or ≤3.0 x ULN if considered due to Gilbert’s syndrome; ALT and AST ≤ 2.5 x ULN or ≤ 5.0 x ULN if due to leukemic involvement; serum creatinine ≤2.0 mg/dL x the ULN). Hydroxyurea was allowed for subjects with rapidly proliferative disease before the start of study therapy and for the first 2 cycles on therapy. Patients with blood lactate levels >2 mmol/L and/or serum pH <7.35 were excluded.

Patients eligible for the Solid Tumor Trial were males or non-pregnant women with a histologically confirmed advanced solid tumor or lymphoma that was metastatic or unresectable, and for which there was no available therapy likely to convey clinical benefit. Additional eligibility criteria included (1) age 18+ years; (2) having received at least one line of systemic therapy in the advanced/metastatic setting or with a relapsed and/or refractory lymphoma with at least two previous lines of systemic therapy; and (3) were not candidates for high-dose therapy/autologous stem cell transplant.

The demographics and baseline characteristics of all patients in both trials are provided in Supplementary Tables 2–5. For both trials, all patients provided written informed consent before enrollment. Participating patients were not offered financial compensation but were offered reimbursement for parking. Out-of-town patients were also offered reimbursement for 1–2 nights at a nearby hotel.

Trial Design

Full study protocols for both trials are available as Supplementary Information. Both study designs and treatment plans are detailed in Figure 1, Supplementary Table 1. The first IACS 010759 clinical trial (‘AML Trial’) was a first-in-human, open-label, phase I study that used a 3+3 dose escalation design. This design enabled expansion of the number of patients per cohort beyond three at each dose level during the dose-escalation phase, to better assess the PK and PD profiles of IACS-010759. The dose of 0.5 mg was selected as the starting dose in this first-in-human AML trial, which corresponded to no more than 1/10th of the level where no adverse effects were observed during pre-clinical toxicology studies. The initial dose of IACS-010759 for cohort 1 and 2 were 0.5 and 1.0 mg, respectively. For the first two cohorts, cycle 1 consisted of a single dose on Day 1 followed by a single dose on Day 8, and subsequent cycles consisted of daily doses. By contrast, cohorts 3 and 4 had induction doses of 2.0 mg and 2.5 mg, respectively, daily for Days 1–7, followed by a maintenance dose of 0.5 mg once per week and 1.0 mg thrice per week, respectively. For both cohorts, the maintenance dose was continued unless suspension or stopping criteria were met.

The second IACS-010759 clinical trial (‘Solid Tumor Trial’) was designed as an open-label phase 1 study. Each cycle comprised of 21 days and consisted of an initial induction phase in cycle 1 followed by a maintenance phase. During the induction phase, IACS-010759 was administered daily for the first 5 or 7 days depending on the dosing cohort under assessment. During the maintenance phase, IACS-010759 was administered with either once-a-week or twice-a-week doses. Dose escalation decisions were determined by the clinical trial Safety Monitoring Committee (SMC) based on toxicity, PK, PD, and antitumor activity. The doses and schedules of the induction and maintenance phases (Figure 1, Supplementary Table 1) were as follows: Cohort 1 received 2.0 mg QD of IACS-010759 for 7 days (induction), followed by 0.5 mg of IACS-010759 once-a-week (maintenance); Cohort 2: 2.5 mg QD of IACS-010759 for 7 days (induction), followed by 1.0 mg of IACS-010759 once-a-week (maintenance); Cohort 3: 3 mgs QD of IACS-010759 for 7 days (induction), followed by 3 mg of IACS-010759 once-a-week (maintenance); Cohort 4: 2.5 mg QD of IACS-010759 for 7 days (induction), followed by 2.5 mg of IACS-010759 twice-a-week maintenance; Cohort 5: 2.0 mgs QD of IACS-010759 for 7 days (induction), followed by 2.0 mg of IACS-010759 twice-a-week (maintenance); Cohort 6: 1.5 mg QD of IACS-010759 for 5 days (induction), followed by 1.5 mg of IACS-010759 twice per week (maintenance).

Study Oversight

The Scientific Review Committee and Institutional Review Board at the University of Texas MD Anderson Cancer Center approved all protocols and amendments for the AML and Solid Tumor Trials, respectively. Both trials were conducted in accordance with the Declaration of Helsinki and the International Council for Harmonization Good Clinical Practice guidelines as well as underwent US Food and Drug Administration safety review (AML – IND #128639; Solid tumor – IND #136859). Because IACS-010759 will not be further developed, both INDs were closed after submission of clinical study reports to the FDA. All adverse events were coded using MedDRA (version 19.0), and classified by system-organ-class (SOC) and preferred term (PT). The Medical Dictionary for Regulatory Activities terminology is the international medical terminology developed under the auspices of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The contract research organization (CRO), Prometrika, conducted all monitoring oversight, including the management and monitoring of the clinical trials (e.g. data management), evaluation of safety adverse events, preparation of safety reports and maintenance of safety database, shipment of study drug, and the collection of investigator documents. All authors contributed to data analysis and interpretation as well as to the preparation of the manuscript. A scientific writer employed by The University of Texas M.D. Anderson Cancer Center provided drafting and editorial assistance for the manuscript. Overall, all authors can confirm the accuracy and completeness of the study presented here.

Trial objectives and endpoints

The primary objective of the AML and Solid Tumor Trial was to determine the safety, tolerability, maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of IACS-010759. For both trials, secondary objectives were to evaluate the PK and preliminary antitumor activity, including overall response rate and duration of response. The exploratory objectives were to evaluate PD and any exploratory predictive biomarkers of IACS-010759 activity.

Safety and tolerability assessments

Patients were diagnosed clinically and managed jointly by the treating oncologists and neurologists at MD Anderson Cancer Center, with electrodiagnostic studies (for example electromyography and nerve biopsies) conducted where clinically indicated. For both AML and Solid Tumor Trials, TEAEs and TRAEs were graded for severity according to the National Cancer Institute CTCAE v4.03. Given the potential for mechanistic toxicity of lactic acidosis during the AML trial, close monitoring of lactate, blood pH, and associated electrolytes was conducted in all patients. Any patient that presented with lactate levels >4 mmol/L, blood pH <7.30, and symptoms attributable to lactic acidosis (e.g. nausea, vomiting, generalized muscle weakness, tachycardia, hypotension, and rapid breathing) was hospitalized for closed monitoring for a minimum of 24 hours. However, patients who presented with lactate levels >4.0 mmol/L, blood pH <7.35, CO2 levels less than the lower limit of normal, an elevated anion gap, and/or symptoms of lactic acidosis were considered to have reached the dose-limiting toxicity upon confirmation of the results by arterial blood sampling. To reduce the variability of pH and lactate measurements, arterial blood gas and venous blood samples were immediately placed in an ice bath and all laboratory tests were performed within 30 minutes after samples were drawn. Upon completion of the venous blood test, any sample showing lactic acidosis had to be confirmed by arterial blood gas, including lactate results.

Determination of human plasma concentrations of IACS-010759

To measure IACS-10759 concentration in human plasma, aliquots of human plasma (50 μL) were first mixed with acetonitrile (200 μL) containing diclofenac as the internal standard (IS). This suspension was vortexed for 10 min and centrifuged at 2000 g for 10 min, after which 100 μL of the extract was aliquoted and diluted with 100 μL of water prior to LC-MS/MS analysis. The UHPLC-MS/MS system consisted of a Shimadzu Nexara UHPLC system coupled with a Sciex 5500 triple quadrupole mass spectrometer operated at the positive mode (ESI+). The optimized source parameters were gas1, 35 psi; gas2, 50 psi; curtain gas, 35 psi; source temperature, 400°C; ion spray voltage, 5500 V. The separation of IACS-010759 was achieved on a Phenomenex Gemini C18 column (5μm, 110Å, 50×2.0mm²) using a gradient mobile phase. Mobile phase A was acetonitrile and water (100/900, v/v) and mobile phase B was acetonitrile and methanol (500/500, v/v) containing 0.2% (v/v) formic acid. The LC gradient (%B) was 22% (0–0.3 min), 22–95% (0.3–1.3 min), 95% (1.3–2.3 min), 95–22% (2.3–2.31 min), and 22% B (2.31–3.0 min). Column temperature was set at 40 °C. Injection volume was 2 μL. IACS-10759 and IS were detected by a multiple reaction monitoring transition m/z 563.1 > 172.1 and m/z 295.9 > 151.2, respectively. Under these conditions, retention time was 1.52 min for IACS-010759 and 1.42 min for IS. The complete analytical run time was 5.0 min. This method was validated over the concentration range of 0.05 to 50.0 ng/mL of IACS-10759 in untreated human plasma.

AML blast collection, OCR analyses, mitochondrial DNA analyses

Fresh blood was obtained in BD Vacutainer^™ Glass Blood Collection Tubes with Sodium Heparin ((Thermo Fisher Scientific: 026853B) and combined into a 50ml falcon tube to which equal volume of Easy Sep Buffer (StemCell Technologies: 20144) was added. This diluted blood was added to a Sepmate tube (StemCell Technologies, Catalog# 85450) containing 25 ml of Lymphoprep (StemCell Technologies: 07811). The tubes were centrifuged for 10 min at room temperature at 1200 g. The top layer was collected in a separate tube and, with 50 mL of Easy Sep Buffer added, centrifuged at 750 g for 6 min at room temperature. The supernatant was discarded, 5–15 ml of ammonium chloride solution (StemCell Technologies: 07850) was added to the pellet, and the solution gently rocked on a shaker for 10–15 minutes. Easy Sep Buffer was added after lysis and centrifuged. The pellet was resuspended in optimum volume of Easy Sep buffer and counted on a Cellometer Auto 1000 Bright Field Cell Counter (Nexcelcom Biosciences). PBMC samples were then depleted of CD3/CD19 positive population using the StemCell separation protocol and custom antibody cocktails (StemCell Technologies).

A single cell suspension of PBMCs at a concentration of 1×10⁸ cells/ml was prepared in EasySep Buffer in a round bottomed tube. To this, custom EasySep Human CD3/CD19 cocktail was added at a concentration of 100 μl/ml of cells. The suspension was vortexed for 10 seconds and incubated for 5 min at room temperature. Dextran Rapid Spheres (StemCell Technologies 50100) were then added at a concentration of 125 μl/ml of cells. The suspension was vortexed for 10 sec and incubated for 3 min at room temperature after which the final volume was made up to 2.5 ml and gently vortexed for 10 sec. The tube was then placed into the EasyEights magnet (StemCell Technologies: 18103) to separate for 10 min at room temperature. Using a 2 ml serological pipet, the cells were carefully collected into a new 5 ml round-bottom tube from the non-magnetic side without removing or moving the tube. The new tube was placed into the EasyEights magnet (StemCell Technologies: 18103) to separate for 10 min at room temperature. The cells (AML blasts) were carefully collected after the second round of suspension and an appropriate volume of PBS was added before counting on a Cellometer Auto 1000 Bright Field Cell Counter (Nexcelcom Biosciences). Blasts were immediately used for the Seahorse assay to measure changes in basal and maximal OCR, with the remainder frozen in Bambanker cell-freezing medium (Thermo Fisher Scientific, no. 50999554) before storage in liquid nitrogen. Changes in mitochondrial DNA and genomic DNA were measured and analyzed with methods previously described⁵⁸.

Metabolomic Assays

Intracellular metabolites were extracted and then analyzed using UHPLC-MS/MS, as previously described^59,60. Briefly, intracellular polar metabolites were extracted using a modified Bligh-Dyer procedure. All LC-MS analyses were performed on a Q Exactive Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Scientific) coupled to a Vanquish Horizon UHPLC system (Thermo Scientific). Metabolite separation was performed using a SeQuant ZIC-HILIC 3.5μm, 100 Å, 150 × 2.1 mm column (Millipore Sigma) and a Kinetex C18,2.6μm, 100 Å, 150 × 2.1 mm column (Phenomenex). Pooled quality control samples were acquired every six samples to ensure optimal instrument performance and consistency. Raw files were processed using SIEVE 2.2.0 SP2 (Thermo Scientific), and the integrated peaks were mined against an in-house database of accurate masses and retention times generated using the IROA 300, MS Metabolite Library of Standards (IROA Technologies), as well as accurate masses from the Human Metabolome database⁶¹ and the KEGG database⁶².

mRNA-seq library preparations and sequencing

mRNA libraries were prepared and sequenced according to published methods⁶³. Briefly, starting with 1 μg total RNA, Illumina True-seq v2 (Cat. # RS-122–2001, RS-122–2002, Illumina) kits were used according to manufactures guidelines to prepare the PolyA Captured mRNA library. All primers and reagents were proprietary and were included in this kit for library preparation. Purified mRNAs were then fragmented and converted to cDNA with reverse transcriptase. Resulting cDNAs were converted to double stranded cDNAs and subjected to end-repair, A-tailing, and adapter ligation. Constructed libraries were amplified using 8 cycles of PCR. The NuGEN Ovation RNA-Seq System v2 (Cat. # 7102–32, NuGEN) was used to convert Total RNA (100 ng) to cDNA by following the manufacturer’s protocol (NuGEN, San Carlos, CA). Briefly, sonication with a Covaris S220 instrument (Covaris, Woburn, MA) was used to break NuGEN-amplified doublestranded cDNAs into ~180 base pair (bp) fragments. Fragmented cDNAs were then processed using the automated SPRIworks Fragment Library System (Beckman Coulter, Fullerton, CA). Uniquely indexed NEXTflex adapters (cat # 514104; Bioo Scientific, Austin, TX) were ligated onto each sample to allow for multiplexing. Adapter-ligated libraries were amplified [1 cycle at 98 °C for 45 s; 15 cycles at 98 °C for 15 s, 65 °C for 30 s, and 72 °C for 30 s; 1 cycle at 72 °C for 1 min; a hold at 4 °C] using a KAPA library amplification kit (KAPA Biosystems, Wilmington, MA) and purified with AMPure XP beads (cat #A63881; Beckman Coulter). Pooled libraries (2.8 nM) were then processed using a cBot (Illumina) for cluster generation before sequencing on an Illumina HiSeq 3000 (2 × 76 bp run).

RNA-sequencing analysis

Quality of the sequencing data was studied by use of the package fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Sequences were then aligned to the human reference genome “GRCh38” (Ensembl Release 96 Databases, released on April 10, 2019) and quantified using Quasi-mapping mode by the package “salmon v0.13.1.”⁶⁴, which calculated gene counts and abundances. Abundance data were then normalized by inter-library normalization, trimmed mean of M values (TMM)⁶⁵, which accounts for library size variation between samples of interest. After alignment, only transcript IDs representing genes of standard chromosomes (1 to 22, X, Y, and MT) were kept, and transcript IDs that were not expressed across all samples (both count and abundance = 0) were removed. In addition, transcript IDs with both low counts and low standard deviations (SD), which may increase incidents of false positives, were removed. Further, we discounted all summarized gene counts other than those with raw counts > 2 and SD > 1. Overall, we obtained 106,209 transcript IDs that represented 19,323 unique genes.

All statistical analyses were performed using R version 3.5.1 (https://www.r-project.org/). For analysis gene expression, we used a linear mixed effect model in which patients were used as random effects and treatment were used as the fixed effect. Due to multiple comparisons, the rate of type I errors was adjusted by using a false discovery rate (FDR) cutoff of < 0.05⁶⁶. As such, 1,065 genes were identified to be significantly associated with treatments. An unsupervised hierarchical clustering analysis for which correlation was used as the distance metric and ward was used as the clustering method was employed to display the expression patterns of significant genes. To identify the functionality of genes of interest, we applied gene ontology (GO) enrichment analysis implemented in an R Package “GOsummaries”⁶⁷. To investigate deregulated biological pathways, we downloaded pathways and gene sets from MSigDB (http://www.gsea-msigdb.org/gsea/msigdb/index.jsp) and used an R package “seq2pathway”⁶⁸ to calculate pathway scores. A linear mixed effect model was then used to identify significant pathways.

Preparation of mouse models

To develop AML patient-derived xenograft (PDX) models, 55 adult female adult female mice (NSG and C57BL/6J) (Jackson Laboratory, NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, Stock No: 005557) were injected intravenously with the PDX AML-04030094 model at 0.5 × 10⁵ cells per mouse and then treated accordingly with clinically relevant doses of IACS-010759. Each mouse received exposure to Cesium-radiation at 250 cGY 24 hours prior to receiving the cell injection. Using an 1 ml syringe and 27 g needle, an intravenous injection of 200 μl (a concentration equal to 0.5 × 10⁵ cells) was injected into the lateral tail vein. Mice were housed in microisolator cages, not exceeding five mice per cage, under a 12-h photoperiod (06:00–18:00).

To prepare mice for investigation of treatment-induced peripheral neuropathy, male C57BL/6J mice (no. 000664, Jackson laboratory) were housed in microisolator cages, not exceeding five mice per cage, under a 12-h photoperiod (08:00–20:00).

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of M.D. Anderson Cancer Center and are in accordance with National Institutes of Health (NIH) guidelines for the care and use of animals. All animals were maintained at the animal facility within The University of Texas MD Anderson Cancer Center. Each cage had corn cob bedding (Anderson Bed o’ Cobs, ¼” inch) with free access to chow (Lab Diet, irradiated 5053) and water. All rooms housing mice had restricted access, with temperature maintained at 22 °C and humidity at 50%. All analyses using mouse models were performed by investigators blinded to treatment.

Effects of IACS-010759 on survival in AML PDX models

Investigation into whether IACS-010759 would extend the survival of AML PDX models began when AML PDX mice exhibited a disease burden of 3% circulating in whole blood. Disease burden was determined by collection of approximately 125 μl of whole blood retro-orbitally from ten nonanesthetized mice into EDTA-coated tubes, followed by storage on wet ice for analysis. Forty-five mice were randomized into the following groups (n = 9 per group), each receiving one of the assigned treatments: 1) vehicle (0.5% Methylcellulose), 2) 0.1 mg/kg IACS-010759, 3) 0.3 mg/kg IACS-010759, 4) 1 mg/kg IACS-010759, and 5) 5 mg/kg IACS-010759. Treatment, at 10 ml/kg, was administered by oral gavage once per day for 5 days on and 2 days off.

After 4 days of treatment, pharmacokinetics were assessed at 2 and 24 hours post-dose. Whole blood (~125 μl) was collected retro-orbitally from each nonanesthetized mouse by placing a heparin lined capillary tube into the lateral canthus and slightly twisting the tube to cause blood to flow directly into EDTA-coated collection vials. Vials were then placed into a centrifuge for 5 minutes at 5000 g. Plasma was isolated using a Pipetman, transferred into a microfuge tube, then placed on dry ice. For long-term storage, samples were stored at −80 °C until analysis.

To measure the survival of treated mice, body weights were collected twice per week. Body condition was assessed when each mouse received its scheduled dose of compound. Disease progression in mice was indicated by loss of body weight, onset of hind-limb paralysis, lethargy, and hunched postures. A mouse was euthanized upon losing >20% of its original body weight or developing hind-limb paralysis. Immediately after euthanization, the spleen and plasma were collected and stored for future analyses.

Behavioral studies assessing treatment-induced peripheral neuropathy

To investigate the effects of treatment-induced peripheral neuropathy, male mice were randomized into groups (n= 4–8/group) that received either IACS-010759 or vehicle (control). Depending on the experiment, mice received either 0.1, 0.3, 1.0, or 5.0 mg of IACS-010759 in 0.5% methyl cellulose or vehicle (control). To assess whether coadministration of the HDAC6 inhibitor ACY-1215 (Ricolinostat, Regenacy Pharmaceuticals) could mitigate treatment-induced neuropathy, mice were randomized into groups that received either IACS-010759 plus ACY-1215, IACS-010759 alone, ACY-1215 alone or vehicle (control) alone. When applicable, IACS-010759 was administered by oral gavage. When applicable, ACY-1215 (Ricolinostat) in 10% DMSO, 30% propylene glycol (#P4347, Sigma), and 60% poly(ethylene glycol)-300 (#81162, Sigma) was administered orally at a dose of 30 mg/kg 60 minutes before each dose of IACS-010759. All treatments in both experiments were administered by oral gavage once per day, 5 days on, 2 days off, followed by a second round of 5 days on.

Mechanical allodynia was quantified using von Frey calibrated filaments at Day 6 after the first dose and then for every 3 days for 24 days. The mechanical stimulus inducing 50% likelihood of withdrawal was determined using the ‘up-down’ calculation method as previously described^69–71. Briefly, mice were habituated on a wire-grid panel for 20 min before testing. A series of von Frey filaments (0.07, 0.16, 0.40, 0.60, 1.00 and 1.40 g, Stoelting) was applied to the plantar surface of the hind paw. Rapid withdrawal or flinching of the hind paw was considered a positive response. In the absence of a response, the next greater force was applied.

Spontaneous pain was measured using a conditioned place preference (CPP) paradigm with retigabine as the conditioned stimulus at two weeks after the last dose of treatment or vehicle (Extended Data Fig. 9a)⁷². At least one week before the CPP procedure, mice were housed in a reversed light/dark cycle (lights off at 08:00). The CPP apparatus consisted of 2 chambers (18 × 20 cm², one dark, one bright) connected by a 15 cm hallway (Stoelting, Wood Dale, IL). On Day 1, each mouse freely explored the apparatus for 15 min. Over the next four days, mice were conditioned to associate the light chamber with pain relief. In the morning, mice were injected intraperitoneally with PBS, rested for 10 min, placed individually in the dark chamber for 20 min, and returned to their respective cages. Three hours later, mice were injected intraperitoneally with 10 mg kg-1 of the retigabine and placed in the bright chamber. On Day 6, drug-free mice explored the apparatus for 15 min. All tests were recorded by video. Change in the time spent in the bright (previously analgesic-paired) chamber at Day 6 as compared with that during Day 1 was quantified on video recordings. Increased time in the analgesic-paired chamber is interpreted as evidence of spontaneous pain.

Sensorimotor function was assessed by a beam-walking test after completion of the CPP test. On Day 1, mice were trained to cross a rectangular beam (85 cm in length with a flat surface of 1.2 cm in width) resting on two poles and elevated 40 cm above the table top. Over 3 consecutive trials, mice were placed at one end of the beam and were trained to cross to the escape platform on the other end. On Day 2, the time needed to cross the 1.2 cm beam was recorded in two trials. On the same day, mice were trained to cross a narrow rectangular beam (85 cm in length, 0.6 cm in width). On Day 3, the time required to cross the narrow rectangular beam was recorded in two trials. On the same day, mice were trained to cross a round beam (diameter 1.2 cm) until mice in the control group could cross the beam successfully in two consecutive trials. On the last day, the the time required to cross the round beam was recorded in two trials. All trials were taped, and the the time required to cross was analyzed by an investigator blinded to treatment and group. The beams and platform were cleaned with 70% ethanol before testing of each new animal.

Mitochondrial function of DRG neurons

Mice were sacrificed by CO2 overdose and DRG neurons were collected from each mouse 3–4 hours after the last dose or 5 weeks after the last dose of either IACS-010759 or vehicle. Mitochondrial bioenergetics of DRG neurons were measured with the XF24 Flux Analyzer (Agilent Technologies Inc, Santa Clara, CA) as previously described⁷¹. Briefly, DRG neurons were plated in XF24 microplates and cultured overnight in Ham’s F-10 medium (#10–070-CV, Corning, NY) with N2 supplement (#17502001, Thermo Fisher Scientific, Waltham, MA). On the day of the assay, medium was changed to XF medium supplemented with 5 mM glucose, 0.5 mM sodium pyruvate, and 1 mM glutamine (Agilent Technologies Inc) for OCR measurement. Oligomycin A (2 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 4 μM), as well as rotenone and antimycin A (2 μM each) (Sigma-Aldrich) were used to determine mitochondrial respiratory properties. The results were normalized to the protein contents of each well.

Immunofluorescence

To quantify IENF, glabrous hind paw skin was collected 12 days after the final dose and processed as described previously73. Briefly, frozen sections (25 μm) were incubated with rabbit anti-PGP9.5 (Abcam, #ab108986, 1:500) and goat anti-collagen IV antibodies (Southern Biotech, #1340–01, 1:100) followed by Alexa-594 donkey anti-rabbit (Thermo Fisher Scientific, #A-21207, 1:500) and Alexa-488 donkey anti-goat secondary antibodies (Thermo Fisher Scientific, #A-11055, 1:500). Images were captured with a Leica SPE confocal microscope (Leica Microsystems, Buffalo Grove, IL). IENF density was quantified as the total number of PGP9.5 stained nerve fibers that crossed the collagen-stained dermal-epidermal junction/length of epidermis (IENFs/mm). Eight mice from each group were measured, with five random images quantified from each mouse for individual IENF counts. Samples were then collected for ATF3 expression analysis 12 days after the last dose. Frozen sections (14 μm) of PFA-fixed, OCT embedded DRG were incubated overnight with anti-ATF3 antibody (Novus Biologicals #NBP-1–85816; 1:500) in PBS with 2% normal donkey serum and 0.1% saponin (Sigma, #S7900) followed by Alexa-fluo 594 donkey anti-rabbit antibody (A-21207) and DAPI (Sigma, #D9542). ATF3 staining was visualized using a Leica SPE confocal microscope. DRG samples from mice that underwent spared nerve injury 2 weeks earlier served as positive controls.

Transmission electron microscopy (TEM) analysis of myelin integrity

For TEM analysis of myelin integrity, mice were euthanized with CO₂ and transcardially perfused with cold PBS at 5 weeks after the last treatment dose. Each sciatic nerve was fixed in 2% glutaraldehyde (EM grade, #16220, Electron Microscopy Sciences) plus 2% PFA (EM grade, #15710, Electron Microscopy Sciences) in PBS for at least 24 hours. Fixed sampled were processed at the High Resolution Electron Microscopy Facility at M. D. Anderson Cancer Center. Briefly, samples were washed in 0.1 M sodium cacodylate buffer and treated with cacodylate buffered tannic acid, post-fixed with 1% buffered osmium and stained en bloc with 0.1% Millipore-filtered uranyl acetate. Samples were then dehydrated in increasing concentrations of ethanol and infiltrated, and embedded in LX-112 medium. Samples were then polymerized in 60 °C over approximately 3 days. Ultrathin sections were cut using a Leica Ultracut microtome and then stained with uranyl acetate and lead citrate in a Leica EM Stainer. Stained samples were examined in a JEM 1010 transmission electron microscope (JEOL USA, Inc, Peabody, MA) using an accelerating voltage of 80 kV. Digital images were obtained using an AMT imaging system (Advanced Microscopy Techniques Corp., Danvers, MA).

Percentage of damaged myelin was quantified as (number of axons with decompacted/loosened myelin/total number of myelinated axons) × 100. For gauging of myelin thickness, maximum diameter was used. The g ratio was quantified as the ratio of axonal/axonal+myelin diameter.

Statistical analysis

Continuous variable summaries, including mean, standard deviation, median, and ranges (minimum and maximum), as well as frequencies and percentages were calculated using SAS Version 9.4 for Windows. If values were missing within patient datasets, the number missing is presented without a percentage. Unless otherwise explicitly specified, missing data were not imputed. The full analysis set included any subject who received any amount of study drug and had at least one on-treatment evaluation. Subject disposition, demographics, baseline characteristics, and study drug exposure are presented by descriptive statistics for each cohort and for the overall trial. All adverse events were coded using MedDRA (version 19.0), and classified by system-organ-class (SOC) and preferred term (PT). The Medical Dictionary for Regulatory Activities terminology is the international medical terminology developed under the auspices of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). Two-way analysis of variance (ANOVA), one-way ANOVA, paired or unpaired Student’s t-test, Chi-square test, Pearson’s correlation, simple linear regression, or Fisher’s exact test were used to calculate statistical significance when appropriate, and were performed in GraphPad 9.0 (San Diego, CA). P<0.05 was considered statistically significant. Analysis of transcriptomic data is described in RNA-seq analysis.

Extended Data

Extended Data Fig. 1. Effect of IACS-010759 on venous lactate and blood pH.

a-d, Relationship between venous lactate and IACS-010759 concentrations in AML (a) cohort 1, (b) cohort 2, (c) cohort 3, and (d) cohort 4. Dotted line indicates 8 nM of plasma IACS-010759. e-h, Relationship between blood pH and plasma IACS-010759 concentrations in AML (e) cohort 1, (f) cohort 2, (g) cohort 3, and (h) cohort 4. Dotted line indicates 8 nM of plasma IACS-010759. i-m, Relationship between venous lactate and IACS-010759 concentrations in Solid Tumor (i) cohort 1, (j) cohort 2, (k) cohort 3, (l) cohort 4, and (m) cohort 5. Dotted line indicates 8 nM of plasma IACS-010759. n-r, Relationship between blood pH and plasma IACS-010759 concentrations in Solid Tumor (n) cohort 1, (o) cohort 2, (p) cohort 3, (q) cohort 4, and (r) cohort 5. Dotted line indicates 8 nM of plasma IACS-010759.

Extended Data Fig. 2. Treatment-induced peripheral neuropathy.

a, b, (a) Ultrastructural examination and (b) electronic microscopy analysis of a biopsy collected from the left superficial peroneal upper leg nerve root of a Cohort 4 patient from the Solid Tumor trial. The patient developed Grade 3–4 peripheral neuropathy while on a treatment regimen of 2.5 mg of IACS-010759 daily during the induction phase (Day 1–7), and 2.5 mg bi-weekly during the maintenance phase. Images indicate severe vacuolar changes in myelin sheath with axonal degenerative changes and atrophy.

Extended Data Fig. 3. Pharmacokinetics of IACS-010759 in AML and Solid Tumor cohorts.

Dosing regimens are detailed in Fig. 1, Supplementary Table 1. a, b, Plasma IACS-010759 concentrations over time in (a) AML Cohort 1 (blue) and Cohort 2 (red); (b) AML Cohort 3 (green) and 4 (purple). c, d, Plasma IACS-010759 concentrations (nM) over time in Solid Tumor (c) Cohort 1 (blue), Cohort 2 (red), and Cohort 3 (green); (d) Cohort 4 (purple), Cohort 5 (orange), and Cohort 6 (black). Each dot represents the mean plasma IACS-010759 concentration at one collection point for one patient.

Extended Data Figure 4. Plasma IACS-010759 concentrations in individual patients.

(a-b) Plasma IACS-010759 concentrations AML patients in (a) cohorts 1 (red) and 2 (blue), which received once daily (QD) dosing, and in (b) cohorts 3–4, which each received an induction and maintenance phase. (e-f) Plasma IACS-010759 concentrations in Solid Tumor (c) cohorts 1 and 5, (d) cohorts 2 and 4, (e) cohort 3, and (f) cohort 6. All cohorts received induction and maintenance phases.

Extended Data Figure 5. Correlations between plasma IACS-010759 concentration and baseline oxygen consumption rate (OCR) from AML blasts.

Baseline OCR has been normalized to cell number. Each symbol represents the mean +/− 95% confidence interval derived from 2–6 technical replicates. Closed circles are predose and open circles indicate post dose values. Blue indicates pre-dose (C1D1). Red indicates after one week of QD dosing (C1D7 - Cohorts 3, 4; C1D14 - Cohorts 1, 2). Black closed circles are other timepoints collected during cycle 1. Correlations analyzed by a Pearson’s correlation coefficient test; p < 0.05. (a) n= 3 at C1D8(6), C1D10; n = 5 at C1D1, C1D1(6) C1D8, and C1D14; n = 6 at all other times. (b) n = 5 at C1D8, C1D8(6), C1D22(6), C1D28, C1D28(6); n = 6 at all other times. (c) n = 6. (d) n = 2 at C1D9, C1D14(6), C1D25(6), C1D28, C1D28(6); n = 3 at C1D8, C1D8(6), C1D14, C1D17, C1D17(6); n = 4 at C1D2, C1D10; n = 5 at C1D1, C1D1(6). (e) n = 5 at C1D10; n = 6 at all other times. (f) n = 4 at C1D8(6); n = 6 at all other times. (g) n = 4 at C1D1(4); n = 5 at C1D1; n = 6 at all other times. (h) n = 6. (i) n = 2 at C1D7(4); n = 3 at C1D7, C1D15, C1D15(4), n = 6 at C1D1, C1D1(4). (j-k) n = 6. (l) n = 3 at C1D1, C1D1(4); n = 4 C1D7, C1D7(4); n = 6 at all other times.

Extended Data Figure 6. Correlations between plasma IACS-010759 concentration and maximal oxygen consumption rate (OCR) from AML blasts.

Maximal OCR has been normalized to cell number. Each symbol represents the mean +/− 95% confidence interval derived from 2–6 technical replicates. Closed circles are predose and open circles are post dose values. Blue indicates pre-dose (C1D1). Red indicates after one week of QD dosing (C1D7 - Cohorts 3, 4; C1D14 - Cohorts 1, 2). Black closed circles are other timepoints collected during cycle 1. Correlations analyzed by a Pearson’s correlation coefficient test; p < 0.05. (a) n = 3 at C1D8(6), C1D10; n = 4 at C1D1, C1D2, C1D8; n = 5 at C1D1(6), C1D14, C1D14(6), n = 6 at all other times. (b) n = 4 at C1D28, C1D28(6); n = 5 at C1D8, C1D8(6), C1C14, C1C14(6), C1D22(6); n = 6 at all other times. (c) n = 6. (d) n = 1 at C1D17; n = 2 at C1D9, C1D14, C1D14(6), C1D25(6), C1D28, C1D28(6); n = 3 at C1D8, C1D8(6), C1D17(6); n = 4 at C1D2, C1D10; n = 5 at C1D1, C1D1(6). (e) n = 4 at C1D10; n = 6 at all other times. (f) n = 4 at C1D8(6); n = 5 at all other times. (g) n = 4 at C1D1(4), C1D15; n = 5 at C1D1, C1D15(4), C1D21, n = 6 at C1D7, C1D7(4). (h), n = 5 at C1D15, C1D15(4); n = 6 at all other times. (i), n = 2 at C1D7(4), C1D15; n = 3 at C1D7, C1D15(4); n = 6 at C1D1, C1D1(4). (j) n = 5 at C1D1, C1D1(4), C1D21, unscheduled collection time point; n = 6 at all other times. (k) n = 3 at C1D15, C1D15(4); n = 6 at all other times. (l) n = 2 at C1D1; n = 3 at C1D1(4); n = 4 at C1D7, C1D7(4); n = 5 at C1D21; n = 6 at C1D15, C1D15(4), end of study.

Extended Data Figure 7. Evidence of target inhibition in AML blasts.

(a-e) Effect of IACS-010759 on levels of (a) NAD+, (b) nicotinamide, (c) tryptophan, (d) glutamine, or (e) alanine in AML blasts from Patients 16, 17, and 19 from AML Cohort 4. Y-axis shows metabolite levels relative to pre-dose levels. Differences from pre-trial levels analyzed by linear regression; *p < 0.05, **p < 0.01, ***p < 0.001. (f-g) Effect of IACS-010759 exposure on NMP, NDP, and NTP levels in AML blasts from (f) Patient 19 and (g) Patient 16 and from AML cohort 4. Y-axis shows nucleotide levels respective to pre-dose levels. Differences from pre-trial levels analyzed by linear regression; *p < 0.05; **p < 0.01, ***p < 0.001.

Extended Data Figure 8. Drug-induced effects on gene expression in AML blasts.

(a) Gene Ontology enrichment analysis on RNA-sequencing (RNA-seq) results from AML blasts collected from Patients 16, 17, and 19 of AML Cohort 4 at several pre- and post-dose timepoints across Cycle 1. (b) Pathway analysis ranking deregulated biological pathways upon IACS-010759 treatment from patients described in (a).

Extended Data Fig. 9. IACS-010759 elevates plasma lactate as well as induces behavioral and physiological changes indicative of peripheral neuropathy in preclinical models.

(a) Effect of escalating doses of IACS-010759 or vehicle on plasma lactate in NSG and B6 mice. n = 4 or n = 5 biologically independent samples from vehicle-treated mice or all other groups, respectively. Data analyzed by two-tailed unpaired Student’s T-test; n.s. = non-significant. Mean ± SE shown. (b) Schematic of the Conditioned Place Preference (CPP) Test. (c) Spontaneous pain assessed with a CPP test (b) after the last dose of IACS-010759 (n = 6) or vehicle (n = 6). Data analyzed by two-sided unpaired Student’s T-test. Mean ± SE shown. (d) Sensorimotor function of mice in (c) assessed with a beam walk test. n = 12 biologically independent samples. Data analyzed with a two-way ANOVA with Tukey’s multiple comparison test. Mean ± SE shown. (e) Oxygen consumption rate (OCR) in the dorsal root ganglion (DRG) from mice in (c) measured under basal conditions (Basal) and after addition of oligomycin (ATP, H + leak), FCCP (Max), or actinomycin + rotenone (spare capacity).; n = 8, n = 4, or n = 8 biologically independent mice for 0 (vehicle), 0.3, or 1 mg/kg IACS-010759, respectively. Data analyzed by two-way ANOVA with Dunnett’s Multiple comparison test. Mean ± SE shown. (f) Density of intraepidermal nerve fibers (IENF) from mice in (b) assessed by quantifying PGP9.5 and nerve fibers crossing into the hind paw epidermis per length (mm) of the basement membrane. n = 12, n = 8, or n = 8 biologically independent samples for vehicle, 1 mg/kg, or 5 mg/kg IACS-010759, respectively. Data analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. Mean ± SE shown. (g) Immunohistochemistry (IHC) analysis of DRG ATF3 expression (pink) in of mice treated with vehicle, or 1 mg/kg or 5 mg/kg IACS-010759. Positive control = spare nerve injury (SNI) with ATF3 staining. Scale bar = 167.2 μm. Independently repeated three times with similar results. (h) (top) Representative transmission electron microscopy cross sections of the sciatic nerve from mice in (c) after the last dose of vehicle or IACS-010759. Scale bar=2 μm. (h)(bottom) Effects of 5 mg/kg IACS-010759 (n = 131 axons/4 mice) or vehicle (n = 205 axons/4 mice) on myelin. Differences from vehicle group analyzed by a two-tailed Fisher’s exact test; ***p = 0.0003, **p = 0.0031, *p = 0.0447.

Extended Data Fig. 10. Co-administration of an HDAC inhibitor mitigates the behavioral symptoms of IACS-010759-induced peripheral neuropathy.

a, Spontaneous pain assessed with a Conditioned Place Preference test (Extended Data Fig. 9b) after the last dose of IACS-010759 or vehicle +/- ACY-1215. n = 11, n = 8, or n = 12 biologically independent mice for vehicle, vehicle + ACY-1215, or 1 mg/kg IACS-010759 + vehicle/ACY-1215, respectively. Data were analyzed by a two-way ANOVA. Mean ± SE shown. b, Sensorimotor function of mice in (a) was assessed with a beam walk test after the last dose of IACS-010759 or vehicle +/- ACY-1215. Data represent time to cross the beam. n = 8 or n = 12 biologically independent mice for vehicle + ACY-1215 or all other groups, respectively. Data analyzed by two-way ANOVA with Tukey’s Multiple comparison test. Mean ± SE shown. (c) (left) Representative transmission electron microscopy cross sections of the sciatic nerve from mice in (a) after the last dose of vehicle (top), IACS-010759 (center), or IACS-010759 + ACY-1215 (bottom). Scale bar=2 μm. (Right) Comparison of effects induced by vehicle (n = 184 axons/4 mice), 0.3 mg/kg IACS-010759 (n = 233 axons/4 mice), 1 mg/kg IACS 010759 (n = 158 axons/4 mice), 1 mg/kg IACS-010759 + ACY1215 (n = 201 axons/4 mice), or ACY-1215 (n = 255 axons/4 mice) on myelin. Analysis by two-tailed Fisher’s exact test; **p = 0.0016 vs vehicle; ##p = 0.0041 vs 1 mg/kg IACS-010759.

Data availability

Requests for access to patient-level data from these trials should be made to the corresponding authors. For each request, an independent review panel at MD Anderson Cancer Center will convene within 30 days of the request and decide whether or not the data will be provided. The data will then be available for up to 12 months. Source data are available for Fig 1; Fig 3, Fig 4a–d, i–k; Fig 5; Extended Data Fig 2, 4–10.