Histidine is selectively required for the growth of Myc-dependent dedifferentiation tumours in the Drosophila CNS

Abstract

Rewired metabolism of glutamine in cancer has been well documented, but less is known about other amino acids such as histidine. Here, we use Drosophila cancer models to show that decreasing the concentration of histidine in the diet strongly inhibits the growth of mutant clones induced by loss of Nerfin-1 or gain of Notch activity. In contrast, changes in dietary histidine have much less effect on the growth of wildtype neural stem cells and Prospero neural tumours. The reliance of tumours on dietary histidine and also on histidine decarboxylase (Hdc) depends upon their growth requirement for Myc. We demonstrate that Myc overexpression in nerfin-1 tumours is sufficient to switch their mode of growth from histidine/Hdc sensitive to resistant. This study suggests that perturbations in histidine metabolism selectively target neural tumours that grow via a dedifferentiation process involving large cell size increases driven by Myc.

Keywords: Drosophila; dedifferentiation; histidine; metabolism; neuroblast.

Figure 1. Dietary histidine withdrawal selectively reduces nerfin‐1 and N^ACT but not pros clonal growth

1. A

   Schematic depicting wildtype NBs (left panel) which express Mira (red), undergoing asymmetric division to generate a GMC, which divides only once to give rise to two postmitotic neurons. The cell fate determinant Pros (blue) is inherited by the GMC, where it translocates to the nucleus to promote differentiation. Postmitotic neurons express Pros (blue) and Nerfin‐1 (green). Upon the loss of Nerfin‐1 (nerfin‐1 ⁻ , middle panel), neurons undergo stepwise reversion, by increasing cellular growth, and switching on stem cell genes while maintaining the expression of neuronal‐specific markers such as Pros, before their complete reversion to NBs, giving rise to clones consisting a mixture of NBs and neurons. In pros ⁻ clones, GMCs fail to differentiate and revert to NBs, giving rise to clones consisting mostly of Mira⁺ NBs. Schematic depicting type II wildtype NB lineages (left) and dedifferentiation of INP to NBs upon Notch overactivation, which gives rise to clones consisting of mostly NBs (right).

2. B–E

   Representative pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n = 13, 14, 13), scale bar = 100 μm.

3. F–H

   Representative pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu) and significantly increased by dietary histidine depletion (25% his), quantified in (F) (n = 28, 22, 7), scale bar = 50 μm

4. I–K

   Representative pictures showing that adult nerfin‐1 clonal growth was significantly reduced on −his diet compared to CDD (measured at day 0, and day 9) quantified in (K) (n = 27, 110, 136). Scale bar = 75 μm. ***P < 0.001

5. L–N

   Representative pictures showing that larval nerfin‐1 clonal growth was significantly reduced on 25% his diet compared to CDD (after 6 days), quantified in (N) (n = 53, 36). Scale bar = 50 μm.

6. O–Q

   Representative pictures showing that adult pros clonal growth was not significantly altered on −his diet compared to CDD (after 7 days), quantified in (Q) (n = 12, 23, 16). Scale bar = 75 μm.

7. R–T

   Representative pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction (25% his) compared to CDD (after 6 days), quantified in (T) (n = 19, 13). Scale bar = 50 μm.

8. U–W

   Representative pictures showing that the growth of larval type II lineages overexpressing N^ACT significantly reduced dietary histidine reduction (25% his) compared to CDD, quantified in (W) (n = 27, 19). Scale bar = 100 μm.

Data information: In all graphs, the key indicates that green bars represent a significant increase (P < 0.05), red bars a significant decrease (P < 0.05), and grey bars no significant change (P > 0.05) in t‐tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC, fold change. See also Figs EV1 and EV4.

Figure EV1. The effect of nutrient restriction and EAA withdrawal on nerfin‐1 mutant clone growth (related to Fig 1)

1. nerfin‐1 clones were induced at 48 h ALH and transferred to standard Drosophila media or agarose/PBS (nutrient restriction, NR) for 3 days, and clone size is reduced (though not significantly, P = 0.054) under NR (n = 90, 116).

2. Schematic depicting heat shock and dissection regimes for larval and adult EAA dietary manipulations.

3. Volume (fold change) of the sum of GFP⁺ nerfin‐1 clones per CNS in animals where clones were induced at 48 h ALH for 1 h and evaluated after 9 days of feeding on CDD or CDD‐EAAs (n = 17, 3, 8, 5, 12, 5, 10, 10, 17, 9).

4. w ¹¹⁸ adults (left, female, right, male) were fed a −his diet labelled with bromophenol blue; blue food was detected in the gut after 3 days of feeding.

5. Larval nerfin‐1, pros and N ^ACT clone volume, clones were induced at 48 h ALH and measured at 6 days ALH (n = 26, 11, 16). pros and N ^ACT are of comparable volume, ns = not significant.

Data information: In all graphs, the key indicates that green bars represent a significant increase (P < 0.05), red bars a significant decrease (P < 0.05), and grey bars no significant change (P > 0.05) in t‐tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC, fold change.

Figure 2. Histidine metabolic perturbations affect nerfin‐1 neuron‐to‐NB reversion

1. Schematic depicting the metabolic reaction between histidine and its downstream metabolites histamine and 3‐methyl‐L‐histidine.

2. Concentration of histidine and 3‐methyl‐L‐histidine from whole Drosophila adults extracts measured by ¹H NMR after 3 days of feeding on either CDD or −his diet (n = 3, 3). Both compounds are reduced to below detection levels (< 0.3 mM) upon dietary his depletion.

3. Dietary supplementation of 3‐methyl‐L‐his for 4 days significantly increased adult nerfin‐1 clonal growth and rescued nerfin‐1 growth inhibition due to his dietary withdrawal (n = 18, 14, 7, 9).

4. Dietary supplementation of histamine does not significantly increase adult nerfin‐1 clone size, but can rescue nerfin‐1 clonal growth inhibition due to his dietary withdrawal (n = 42, 14, 23, 25).

5. Larval nerfin‐1 clonal growth is significantly decreased upon overexpression of HDC RNAi (n = 23, 36).

6. Schematic depicting stepwise neurons to NBs reversion in nerfin‐1 type I lineages where neurons express Elav, NBs express Dpn, and reverting neurons express both markers.

7. Hdc inhibition significantly reduced the percentage of Dpn⁺ NBs (n = 16, 17) and significantly increased the percentage of Elav⁺ neurons per clone (n = 16, 17).

8. Hdc^Ri overexpression reduced cellular growth of nerfin‐1 NBs (n = 17, 19) and neurons (n = 36, 19) measured as the ratio of nucleolus/nuclear volume.

Data information: In all graphs, the key indicates that green bars represent a significant increase (P < 0.05), red bars a significant decrease (P < 0.05), and grey bars no significant change (P > 0.05) in t‐tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC, fold change. See also Figs EV2 and EV3.

Figure EV2. ¹H NMR can detect changes in dietary histidine and his dietary manipulation differentially affects nerfin‐1 ⁻ and pros ⁻ clonal growth (related to Fig 2)

1. Spectra showing a region from 8.0 to 7.1 ppm from extracted whole adult fly polar metabolome before (upper panel) and after (lower panel) clearance of dietary histidine from the gut (see Materials and Methods), boxed panel highlights the histidine peak. Replica peaks for histidine are seen in the boxed areas at ˜7.9 (ε‐proton) and 7.1 (δ‐proton) ppm in histidine‐replete: “+” (0.5 g/l his) and “2x” (1.0 g/l his) CDD profiles; histidine peaks are not seen in “−” (0 g/l his) profiles in either panel.

2. nerfin‐1 ⁻ clone volume is increased by ˜4‐fold upon feeding on 2× his (n = 24, 15).

3. pros ⁻ clone volume did not significantly changed upon feeding on 2× his (n = 19, 34).

Data information: In all graphs, the key indicates that green bars represent a significant increase (P < 0.05), red bars a significant decrease (P < 0.05), and grey bars no significant change (P > 0.05) in t‐tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC, fold change.

Figure EV3. The effect of manipulating histidine metabolic pathway intermediates on control and nerfin‐1 clones (related to Figs 3 and 4)

1. A

   500 mg/ml of the histamine receptor inhibitor cimetidine (n = 39, 23) did not significantly alter nerfin‐1 clonal growth in the adult CNS.

2. B

   Knockdown of histamine inhibitor HisCl1 (HisCl1^Ri), knockdown verified in Oh et al, 2013, did not significantly alter nerfin‐1 clonal growth in the larval CNS (n = 37, 42).

3. C

   Hdc knockdown did not significantly alter the growth of wildtype larval CNS clones (n = 42, 36).

4. D

   Histamine supplementation did not significantly increase nerfin‐1 clone size, but significantly rescued nerfin‐1;HdcRi clonal growth in the larval CNS (n = 15, 10, 12, 9).

5. E

   Hdc knockdown did not significantly alter the amount of cell death in nerfin‐1 clones in the larvae (n = 43, 35).

6. F

   The speed of the cell cycle in fully dedifferentiated NBs > 8 μm (n = 11, 9) and dedifferentiating neurons < 8 μm (n = 11, 9) was not significantly altered by Hdc inhibition in nerfin‐1 larval clones.

7. G–I

   Representative pictures showing that Hdc inhibition significantly reduced the % of Myc⁺ NBs (red) and Myc⁺ Elav⁺ (blue) neurons in nerfin‐1 larval clones (G–H’’), quantified in (I) (n = 14, 14, 13, 16). Scale bar = 50 μm.

Data information: In all graphs, the key indicates that green bars represent a significant increase (P < 0.05), red bars a significant decrease (P < 0.05), and grey bars no significant change (P > 0.05) in t‐tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC, fold change.

Figure 3. Histidine depletion slows down nerfin‐1 dedifferentiation via decreasing cellular growth

1. A–E

   Representative images of nerfin‐1 clones in the adult VNC after 6 days of feeding on CDD or −his diet, scale bar = 100 μm. Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav⁺ neurons (red) quantified in (D) (n = 16, 17) and significantly fewer Mira⁺ NBs (blue), quantified in (E) (n = 12, 22), without significantly altering the amount of cell death per clone quantified in (C) (n = 6, 7).

2. F, G

   Larval nerfin‐1 clones consisted of significantly greater proportion of differentiated neurons (n = 10, 7) and significantly reduced proportion of NBs per clone upon his dietary depletion (25% his, n = 9, 11).

3. H–P

   Representative images of adult nerfin‐1 (I–J’, green) and pros clones (K–L’, green), scale bar = 5 μm. In nerfin‐1 clones, the size of the NBs (Ase⁺, blue) was significantly reduced upon 6 days of histidine dietary withdrawal, quantified in (H) (n = 33, 20). Cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was also significantly reduced, quantified in (M) (n = 33, 20). In pros clones, the size of the NBs (Ase⁺ blue) and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume were not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (H) (n = 9, 8) and (O) (n = 9, 7). Cellular growth measured as the ratio of nucleolus/nuclear volume was significantly reduced in larval nerfin‐1 NBs after 6 days his dietary reduction (25% his compared to CDD, n = 14, 6) quantified in (N). Panel (P) shows that the larval nerfin‐1 neuron‐to‐NB reversion requires 15‐fold increase in cellular volume (n = 10) and pros GMC‐to‐NB reversion requires approximately 3.5‐fold increase in cellular volume (n = 31).

Data information: In all graphs, the key indicates that green bars represent a significant increase (P < 0.05), red bars a significant decrease (P < 0.05), and grey bars no significant change (P > 0.05) in t‐tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC, fold change. See also Fig EV2.

Figure 4. Histidine deprivation inhibits nerfin‐1 clones via Myc and eIF4E

1. A–D

   Representative pictures showing that pros NBs and GMCs are smaller than wildtype (NBs and GMCs are distinguished at telophase, as a doublet of unequal size), quantified in (C) (n = 25, 31, 21, 16). pros NBs exhibit similar nucleolus/nuclear ratio (white arrows, nucleolus marked by Fib) compared to wildtype and pros;Tor ^DN NBs, quantified in (D) (n = 10, 18, 15).

2. E–I

   Representative pictures showing pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E, F), nucleolus labelled with Fib (red) [quantified in (G) (n = 44, 43)], clone size [quantified in (H) (n = 41, 34)] or the proportion of Dpn⁺ NBs and Elav⁺ neurons per clone [quantified in (I) (n = 15, 15, 15, 15)].

3. J–N

   Representative pictures showing that nerfin‐1 clones (green) generated in myc hypomorphic background (J, K) exhibited significantly reduced nucleolus/nuclear ratio, [nucleolus labelled with Fib (red)] and reduced clone size, quantified in (L) (n = 34, 27) and (M) (n = 106, 75), and displayed an increased proportion of differentiated Elav⁺ neurons and reduced proportion of Dpn⁺ NBs per clone, quantified in (N) (n = 20, 20, 20, 20). scale bar = 10 μm

4. O

   Reducing Myc dosage with the hypomorphic Myc allele, myc ^P0, abolished the increase in nerfin‐1 clone size mediated by 2× histidine dietary intake, 2× his (n = 41, 18, 10, 57).

5. P

   Overexpression of myc restored the growth of nerfin‐1 clones inhibited by histidine dietary reduction, 25% his (n = 51, 36, 39, 29).

6. Q, R

   pros (Q) and nerfin‐1 (R) clones are reduced upon Tor inhibition (n = 22, 63) compared to control (n = 51, 65), but their growth differentially depends on amino acid transporter Slif (n = 12, 32) and downstream growth regulators eIF4E (n = 21, 47) and S6K (n = 39, 37).

Data information: In all graphs, the key indicates that green bars represent a significant increase (P < 0.05), red bars a significant decrease (P < 0.05), and grey bars no significant change (P > 0.05) in t‐tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC, fold change. See also Fig EV3.

Figure EV4. Hdc knockdown reduces N^ACT larval type II CNS clones, but not the growth of N^ACT and scrib ¹ ;Ras ^V12 larval eye imaginal disc clones (related to Figs 1 and 4)

1. A–C

   Representative pictures showing that larval type II N^ACT MARCM clonal growth was significantly reduced by Hdc (A–B’’) and Myc knockdown, quantified in (C) (n = 11, 7, 7). Scale bar = 50 μm.

2. D–G

   Representative pictures showing that Myc (F) and Hdc (E) inhibition was sufficient to significantly reduce N ^ACT clonal growth in the larval eye imaginal epithelia, quantified in (G) (n = 75, 80, 75). Scale bar = 100 μm.

3. H–J

   Hdc inhibition did not significantly alter the growth of scrib ¹ ;Ras ^V12 larval eye imaginal epithelia clones, quantified in (J) (n = 57, 33). Scale bar = 100 μm.

Data information: In all graphs, the key indicates that green bars represent a significant increase (P < 0.05), red bars a significant decrease (P < 0.05), and grey bars no significant change (P > 0.05) in t‐tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC, fold change.